Unparalleled Product Quality, Validation and Technical Support. Catalog of Antibodies for apoptosis

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1 Unparalleled Product Quality, Validation and Technical Support Catalog of Antibodies for apoptosis

2 INNOVATIVE DISCOVERY TOOLS FOR SIGNAL TRANSDUCTION RESEARCH Catalog of Antibodies for the Study of Apoptosis Table of Contents Caspase Signaling and Cleaved Substrates 3 Granzymes and Other Proteases 6 Family Members 6 IAP Family and Mitochondrial Proteins 8 TNFR Family and Adaptor Proteins 11 Autophagy 12 Transcriptional Regulators 14 Reference Pathways Apoptosis Overview 16 Inhibition of Apoptosis 16 Death Receptor Signaling 17 Mitochondrial Control of Apoptosis 17 Select Application References 18 Application and Reactivity Key Applications: W Western IP Immunoprecipitation IHC Immunohistochemistry IHC-P Immunohistochemistry (Paraffin) IHC-F Immunohistochemistry (Frozen) IC Immunocytochemistry IF Immunofluorescence IF-IC Immunofluorescence (Immunocytochemistry) IF-F Immunofluorescence (Frozen) IF-P Immunofluorescence (Paraffin) F Flow Cytometry E ELISA D DELFIA Reactivity: H human M mouse R rat Hm hamster Mk monkey B bovine Dr drosophila All all species expected ( ) 100% sequence homology Cell Signaling Technology (CST) provides the highest possible quality activation-state and total antibodies available for the study of signaling pathways central to cell survival and programmed cell death. CST s antibodies have been extensively validated by our in-house clinical applications group in applications including immunohistochemistry, immunofluorescence, ELISA, flow cytometry and other drug discovery technologies. Technical support is provided by the same scientists who develop and produce the antibodies and know them best. CST s phosphorylation-specific antibodies are the most highly cited and are core reagents in multiple drug discovery platforms. Over the years CST has invested in the development of new antibody production technologies. As a result we are now employing a new and proprietary rabbit monoclonal technology which enables the production of antibodies with even greater sensitivity and specificity. Apoptosis-Related Antibody Sampler Kits CST s Antibody Sampler Kits contain sample size antibody sets organized by signaling pathway or by specific target protein and provide an economical means to test a selection of our best-selling products in your field of interest. Each antibody sampler kit contains enough primary and secondary antibodies to perform four Western mini-blots per target. Sampler Kits Phospho-Akt Pathway Sampler Kit #9916 Akt Isoform Sampler Kit #99 Apoptosis Sampler Kit #9915 Apoptosis Antibody Sampler Kit (Mouse Specific) #99 Pro-Apoptosis Family Antibody Sampler Kit #9942 Pro-Survival Family Antibody Sampler Kit (Human Specific) #9941 Cleaved Caspase Antibody Sampler Kit #9929 Forkhead Signaling Antibody Sampler Kit #9946 Phospho-IKKa/b (Ser176/1) Antibody Sampler Kit #9958 NF-kB Pathway Sampler Kit #9936 NF-kB Non-Canonical Pathway Antibody Sampler Kit #4888 Phospho-p53 Antibody Sampler Kit #9919 mtor Pathway Antibody Sampler Kit #9964 Alexa Fluor is a trademark of Molecular Probes, Inc. / Rabbit Monoclonal Antibodies produced using technology from Epitomics, Inc. under U.S. Patent No. 5,675,063.

3 Caspase Signaling and Cleaved Substrates Apoptosis is a physiological process leading to a highly regulated, programmed form of cell death. Programmed cell death is a normal part of growth and development of multicellular organisms; dysregulation of apoptosis can lead to abnormal development and oncogenesis. This process is controlled by numerous proteins, including a family of cysteine aspartic acid proteases known as caspases. Caspases regulate apoptosis by cleaving target proteins at specific amino acid residues at several steps within a proteolytic cascade. Apoptotic signals trigger caspase activation, a process that involves the cleavage and subsequent rejoining of the large (p) and small (p10) caspase domains and the removal of a variable-length amino-terminal prodomain. Approximately 15 caspases have been identified in mammals, and these can be classified based on their structure, molecular function or substrate preference. For example, caspases with longer prodomains (including caspase-2, -8, -9, -10 and -12) are often referred to as initiator caspases, since they are usually closely coupled to upstream pro-apoptotic signals. Once activated, these initiator caspases will cleave and activate downstream effector or executioner caspases (including caspase-3 and -7), which modify several proteins (i.e. PARP1, a-fodrin, DFFA/DFFB and LMNA) that ultimately promote cell death. Under some conditions, amplification of the apoptotic signal is required to trigger programmed cell death. In addition to controlling cellular apoptosis, caspases (particularly caspase-1, caspase-5 and caspase-11) also play a role in activating immature cytokines during the inflammatory response. Some caspases can be inhibited through direct interactions with members of the inhibitor of apoptosis protein (IAP) family. While some IAP inhibitors simply block caspase activity through their binding, other IAPs act as ubiquitin ligases and target these proteases for the ubiquitin-mediated degradation pathway. Interaction between the IAP and IAP inhibitors (such as Smac/Diablo, XAF1 and HTRA2/OMI) relieves IAP-mediated inhibition of caspases and promotes apoptosis through caspase cleavage and activation. Cytochrome c release from the mitochondrion during apoptosis leads to amplification of the caspase signaling cascade via formation of the apoptosome. A radially-symmetric structure, the apoptosome assembles upon binding of cytochrome c (blue) to Apaf-1 (beige), followed by recruitment of caspase-9 (red). Once assembled, the apoptosome s activated caspase-9 cleaves and activates cytosolic caspase-3 (red, floating) and rapidly amplifies the caspase cascade. This leads to the destruction of numerous intracellular targets by caspase-3 including the actin cytoskeleton (green). For a complete listing of our products see

4 Caspase Signaling Applications Reactivity Apoptosis Sampler Kit #9915 Apoptosis Antibody Sampler Kit (Mouse Specific) #99 Cleaved Caspase Antibody Sampler Kit #9929 Cleaved Caspase-3 (Asp175) Antibody #9661 W, IHC-P, IHC-F, IF-IC, F H, M, R, B Cleaved Caspase-3 (Asp175) Antibody IF-IC, F H, (M, R) (Alexa Fluor 488 Conjugate) #9669 Cleaved Caspase-3 (Asp175) (5A1) Rabbit mab #9664 W, IP, IHC-P, IHC-F, IF-IC, F H, M, R Caspase-3 Antibody #9662 W, IP, IHC-P H, M, R Caspase-3 (8G10) Rabbit mab #9665 W, IP H, M, R Caspase-3 (3G2) Mouse mab #9668 W H Apoptosis Marker: Cleaved Caspase-3 (Asp175) Antibody (Fluorescein Conjugate) #9667 IF-IC H, M, R Apoptosis Marker: Cleaved Caspase-3 (Asp175) Western Detection Kit #96 Apoptosis Marker: SignalStain Cleaved Caspase-3 (Asp175) IHC Detection Kit #81 PathScan Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190 Caspase-3 Control Cell Extracts #9663 Cleaved Caspase-3 (Asp175) Blocking Peptide #10 IHC-P H New SignalSlide Cleaved Caspase-3 (Asp175) IHC Controls #8104 IHC-P H Caspase-1 Antibody #2225 W, IP, IHC-P H Caspase-2 (C2) Mouse mab #2224 W H Cleaved Caspase-6 (Asp162) Antibody #9761 W H, M, R Caspase-6 Antibody #9762 W H, M, R Cleaved Caspase-7 (Asp198) Antibody #9491 W, IP, F H, M, R Caspase-7 Antibody #9492 W, F H, M, R Caspase-7 (C7) Mouse mab (Human Specific) #9494 W H Caspase-7 Activity Assay Kit #98 IP H Cleaved Caspase-8 (Asp384) (11G10) Mouse mab #9748 W H Cleaved Caspase-8 (Asp391) (18C8) Rabbit mab #9496 W, IHC, IF-IC, F H Caspase-8 Antibody (mouse specific) #4927 W M Caspase-8 (1C12) Mouse mab #9746 W, IP H Phospho-Caspase-9 (Thr125) Antibody #2226 W H, (M) Cleaved Caspase-9 (Asp315) Antibody (Human Specific) #95 W, IP H Cleaved Caspase-9 (Asp3) Antibody (Human Specific) #91 W, IP H Cleaved Caspase-9 (Asp353) Antibody (Mouse Specific) #99 W, IF-IC M Cleaved Caspase-9 (Asp353) Antibody (Rat Specific) #97 W R Caspase-9 Antibody (Human Specific) #92 W, F H Caspase-9 Antibody (Mouse Specific) #94 W M Caspase-9 Antibody (Rat Specific) #96 W R Caspase-9 (C9) Mouse mab #98 W H, M, R Caspase-10 Antibody #9752 W H, M, R Caspase-12 Antibody #22 W M Jurkat Apoptosis Cell Lysates (etoposide) #43 Chaps Cell Extract Buffer (10X) #9852 Staurosporine #9953 Tumor Necrosis Factor-a #2169 PathScan Cleaved Caspase-3 (Asp175) Sandwich ELISA Kit #7190 OD 4nm Staurosporine (µm) Cleaved Caspase-3 Full length Caspase-3 Cleaved Caspase Staurosporine (µm) Treatment of HeLa cells with staurosporine stimulates cleavage of caspase-3 protein, detected by #7190 (top figure), but does not affect the level of total caspase-3 protein detected by Western blot (bottom figure), using Cleaved Caspase-3 (Asp175) Antibody #9661 (upper panel) or Caspase-3 Antibody #9662 (lower panel). Caspase-3, the primary executioner of apoptosis, is responsible for most signature events of apoptosis, including DNA degradation, membrane blebbing and nuclear breakdown. Caspase-3 cleaves many target proteins, including (ADP-ribose) polymerase (PARP), cytoskeletal protein a-fodrin, DNA fragmentation inhibitor DFF45, actin-regulatory protein gelsolin, cyclin-dependent kinase inhibitor CDKN1A and p21 activated kinase PAK2. Caspase-3 activation requires proteolytic processing of its zymogen into activated small and large subunits by granzyme B and caspase-9, which cleave and activate caspase-3 through separate but overlapping pathways. Caspase-3 activity is inhibited by phosphorylation of Ser1 by p38 MAPK or by NO-induced S-nitrosylation at its catalytic site. Caspase- 3 function is associated with several human disorders, including Alzheimer and Huntington disease. Cleaved Caspase-8 (Asp391) (18C8) Rabbit mab #9496 Events Cleaved Caspase-8 (Asp391) Flow cytometric analysis of Jurkat cells, untreated (blue) or etoposide-treated (green), using #9496 compared to a nonspecific negative control antibody (red). Apoptosis induced by CD95 (Fas/APO-1) and tumor necrosis factor receptor 1 (TNFR1) activates caspase-8, leading to the release of the active fragments, p18 and p10, of caspase-8. Activated caspase-8 cleaves and activates downstream caspases (i.e. caspase-1, -3, -6 and -7), and inactivates RIPK1 and PAK2 kinases to promote the alternate pathway of cell survival. Caspase-8 deficiency in humans produces unregulated apoptosis and can result in immunodeficiency disorders and cancer. IF analysis of HeLa cells, untreated (top) or treated with cytotoxic anti-fas Antibody (bottom), using #9496. Applications and Reactivity Key, page 2.

5 TNF-a FasL Death stimuli Cleaved Substrates ER Stress [Ca ++ ] Caspase-12 Caspase-6 TRADD P Ser191 FADD P Ser194 Caspase-8/10 FLIP Caspase-2 Apaf-1 Cyto c Caspase-9 IAPs Caspase 3 Lamin A a-fodrin DFF + Cleaved Parp (Asp214) Etoposide Apoptosis FADD RIP CRADD Smac/ HtrA2 Caspase-7 PARP Cleaved PARP (Asp214) Antibody (Human Specific) #9541 WB analysis of Jurkat cells, untreated or etoposide-treated (25 µm), using #9541. Applications Reactivity Acinus Antibody #4934 W, IF-IC H, M, R, Mk Cleaved PARP (Asp214) Antibody (Human Specific) #9541 W, IHC-P, IF-IC, F H Cleaved PARP (Asp214) Antibody (Human Specific) (Fluorescein Conjugate) #9547 IC H Cleaved PARP (Asp214) (19F4) Mouse mab (Human Specific) #9546 W, IF-IC H Cleaved PARP (Asp214) Antibody (Mouse Specific) #9544 W, IC, IF-IC M Cleaved PARP (Asp214) (7C9) Mouse mab (Mouse Specific) #9548 W, IF-IC M Cleaved PARP (Asp214) Antibody (Rat Specific) #9545 W, IC R PARP Antibody #9542 W H, M, R PARP (46D11) Rabbit mab #9532 W, IP, IF-IC H, M, R, Mk PathScan Cleaved PARP (Asp214) Sandwich ELISA Kit #7262 E H, M Cleaved DFF45 (Asp224) Antibody #9731 W H DFF45/DFF35 Antibody #9732 W, IP H Cleaved a-fodrin (Asp1185) Antibody #2121 W H a-fodrin Antibody #2122 W H Cleaved Lamin A (Asp2) Antibody #31 W H, M, R Cleaved Lamin A (Small Subunit) Ab #35 W, IHC-P H, M, R Cleaved Lamin A (Small Subunit) (H5) Mouse mab #36 W, IF-IC H, M, R Lamin A/C Antibody #32 W, IP, IHC-P, IF-IC, F H, M, R, (B) Cleaved IL-1b (Asp116) Antibody #21 W H IL-1b Antibody #22 W H Lamin A/C Antibody # Lamin A/C Confocal IF analysis of HT-29 cells, untreated (left) or staurosporine treated (right), using #9541 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye Cleaved Lamin A/C Staurosporine WB analysis of extracts from HeLa cells, untreated or staurosporine-treated (1 µm), using #32. PARP, a 116 nuclear poly (ADP-ribose) polymerase, appears to be involved in DNA repair predominantly in response to environmental stress. This protein can be cleaved by many ICE-like caspases in vitro, and is one of the main cleavage targets of caspase-3 in vivo. In human PARP, the cleavage occurs between Asp214 and Gly215, which separates PARP s amino-terminal DNA binding domain (24 ) from its carboxyterminal catalytic domain (89 ). PARP is important for cells to maintain their viability; cleavage of PARP facilitates cellular disassembly and serves as a marker of cells undergoing apoptosis. Cleaved Lamin A (Small Subunit) (H5) Mouse mab # HeLa NIH/3T3 C6 Jurkat Staurosporine Etoposide Cleaved Lamin A/C (Small Subunit) WB analysis of extracts from HeLa, NIH/3T3, and C6 cells, untreated or staurosporinetreated (1 µm), and Jurkat cells, untreated or etoposide-treated (25 µm), using #36. IHC analysis of paraffin-embedded human lung carcinoma using #32 in the presence of control peptide (left) or antigen-specific peptide (right). Lamin A is a nuclear membrane protein important in cell cycle control, DNA replication and chromatin organization (1-3). Lamin A is specifically cleaved by caspase-6, with cleaved lamin A serving as a marker for caspase-6 activation. During apoptosis, lamin A (70 ) is cleaved into large ( 45 ) and small (28 ) fragments. The cleavage of lamins results in nuclear dysregulation and cell death. For a complete listing of our products see

6 Granzymes and Other Proteases Granzymes are members of a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer (NK) cells that are key components of immune responses to pathogens and transformed cells. They are synthesized as zymogens and processed into mature enzymes through cleavage of a leader sequence. Granzymes are released by exocytosis in lysosomelike granules containing perforin, a membrane pore-forming protein that facilitates the entry of cytotoxic serine proteases (including granzymes) into target cells. Granzyme B has the strongest apoptotic activity of all the granzymes as it cleaves substrates at aspartic acid residues thereby activating procaspases directly and cleaving downstream caspase substrates. Cathepsin D is a ubiquitously expressed lysosomal aspartyl protease involved in the normal degradation of proteins. It is synthesized as preprocathepsin D that is cleaved into and glycosylated to produce 2 subunits, the heavy and light chains. Cathepsin D may also be secreted into the cytosol during apoptosis and contribute to cleavage of substrates implicated in the apoptotic pathway. Applications Reactivity Cathepsin D Antibody #2284 W, IHC-P, IF-IC, F H, Mk Granzyme A Antibody #4928 W, E H Granzyme B Antibody #4275 W, E H, M, R Perforin Antibody (Mouse Specific) #3693 W, IF-IC, F M Family Members Survival factors PKA p90rsk Ser112 P P Ser155 Caspase-6 Lamin A Bad Caspase-12 Akt P Ser136 IAPs a-fodrin BID FasL FADD P Ser194 Caspase-8 Bad Bcl-xL Caspase 3 DFF Apoptosis tbid Smac/ HtrA2 Bcl-xL Bak Caspase-7 PARP Death stimuli Bax Bax Ser70 P P Thr56 Cyto c Caspase-9 Apaf-1 The family consists of a number of evolutionarily conserved proteins that regulate apoptosis through control of mitochondrial membrane permeability and release of the pro-apoptotic factor, cytochrome c. In general, proteins either work to promote or oppose apoptosis. Family members that favor cell death include those containing multiple BH domains (i.e. Bax, Bak and Bok), and those that contain only the BH3 sequence (including Bad, Bik, Bid, Puma, Bim, Bmf, Noxa and Hrk). Anti-apoptotic proteins contain the founding member of the group,, as well as Bcl-xL, Mcl-1, A1 and Bcl-w. Phospho-Bad (Ser112) (A9) Rabbit mab #5284 Phospho-Bad (Ser112) Bad + TPA WB analysis of extracts from untreated or TPA-treated COS cells, using #5284 (upper), or Bad Antibody #9292 (lower). IHC analysis of paraffin-embedded human breast carcinoma, untreated (left) or l phosphatase treated (right), using #5284. Bad, a proapoptotic family member, promotes cell death by displacing Bax from binding to and Bcl-xL. Survival factors such as IL-3 inhibit Bad s apoptotic activity by activating intracellular signaling pathways resulting in the phosphorylation of Bad at Ser112 and Ser136, which prevents the association between Bad, and Bcl-xl by promoting the binding of Bad to Akt promotes cell survival via its ability to phosphorylate Bad at Ser136. Bad is phosphorylated at Ser112 both in vivo and in vitro by p90rsk and mitochondria-anchored PKA. Phosphorylation of Ser155 in the BH3 domain by PKA plays a critical role in blocking the dimerization of Bad and Bcl-xL. PathScan ELISA Kits #7182 and #7162 Absorbance 4nm Bad (total) Untreated phosphatase-treated TPA-treated Phospho-Bad (Ser112) Treatment of OVCAR8 cells with TPA stimulates phosphorylation of Bad at Ser112, detected by PathScan Phospho-Bad (Ser112) Sandwich ELISA Kit #7182, but does not affect the level of total Bad protein detected by #7162. l phosphatase treatment of control cell lysates (00 U/mL for minutes at 37 C) abolishes the basal phosphorylation of Bad as shown by both Sandwich ELISA and Western analysis. Applications and Reactivity Key, page 2.

7 promotes survival in response to apoptotic stimuli through inhibition of mitochondrial cytochrome c release. It has been implicated in modulating mitochondrial calcium homeostasis and proton flux. Several phosphorylation sites, including Thr56, Ser70, Thr74 and Ser87, may be targets of the ASK1/MKK7/JNK1 pathway, and may be markers for mitotic events. Mutation of at Thr56 or Ser87 inhibits its anti-apoptotic activity during glucocorticoid-induced apoptosis of T lymphocytes. Ile-3 and JNK-induced phosphorylation at Ser70 may be required for its enhanced anti-apoptotic functions. Phospho- (Ser70) (5H2) Rabbit mab #2827 phospho- (Ser70) dna (pi) 1023 Flow cytometric analysis of Jurkat cells, using # Phospho- Paclitaxel phosphatase WB analysis of extracts from Jurkat cells, untreated or treated with paclitaxel (1 μm, overnight) and with or without λ phosphatase, using #2827 (upper) or #2876 (lower). Applications Reactivity Phospho-Bad (Ser112) Antibody #9291 W, IP H, M, R, Mk Phospho-Bad (Ser112) (A9) Rabbit mab #5284 W, IHC-P, F H, Mk Phospho-Bad (Ser112) (7E11) Mouse mab #9296 W, IP, IHC-P H, M, R, Mk Phospho-Bad (Ser155) Antibody #9297 W H, M Phospho-Bad (Ser136) (5D8) Mouse mab #5282 E H New Phospho-Bad (Ser136) (185D10) Rabbit mab #5286 W, IP H, M, Mk Phospho-Bad (Ser136) Antibody #9295 W H, M, R Bad Antibody #9292 W, IP H, M, R, Mk New Bad (11E3) Rabbit mab (IP Preferred) #9268 W, IP H, M, R, (Mk) PathScan Phospho-Bad (Ser112) Sandwich ELISA Kit #7182 E H, M, Mk PathScan Total Bad Sandwich ELISA Kit #7162 E H, Mk Bad Control Proteins #9293 Phospho-Bad (Ser112) Blocking Peptide #1026 New Bak Antibody #3814 W H, M, Mk Bax Antibody #2772 W, IP H, M, R, Mk Bax Antibody (Human Specific) #2774 W, IP, IHC-P, IF-IC, F H Pro-Survival Family Antibody Sampler Kit (Human Specific) #9941 Pro-Apoptosis Family Antibody Sampler Kit #9942 Phospho- (Thr56) Antibody (Human Specific) #2875 W H Phospho- (Ser70) (5H2) Rabbit mab #2827 W, IP, IF-IC, F H Antibody #2876 W H, M, R New (E3) Rabbit mab #2870 W, IP H, M, R, Mk, (C, B) Antibody (Human Specific) #2872 W H Bcl-w (31H4) Rabbit mab #2724 W, IP, F H, M, R Bcl-xL Antibody #2762 W, IP, IHC-P H, M, R, Mk Bcl-xL (54H6) Rabbit mab #2764 W, IP, IHC-P, IHC-F, F H, M, R, Mk New Bcl-xL (54H6) Rabbit mab (Alexa F H, M, R Fluor 488 Conjugate) #2767 Bcl-xL Blocking Peptide #1225 W, IHC-P H, M BID Antibody (Human Specific) #02 W, IP H BID Antibody (Mouse Specific) #03 W M BID (7A3) Mouse mab (Human Specific) #06 W H Bik Antibody #4592 W, IHC-P H, M, R, Mk New Phospho-Bim (Ser69) Antibody #4581 W, IP H, M, R Bim Antibody #2819 W, IP, IF-IC, F H, M, (Mk) Bmf Antibody #4692 W H, M, R, Mk Bok Antibody #4521 W, IP H, M, R, Mk Mcl-1 Antibody #4572 W H Puma Antibody #4976 W, IHC-P, IF-IC, F H, M, R (E3) Rabbit mab #2870 Jurkat C2C12 C6 COS WB analysis of extracts from various cell types using #2870. Bim Antibody #2819 IF analysis of MCF-7 cells using #2819. For a complete listing of our products see 10 MOLT-4 A Raji 293 MCF-7 WB analysis of extracts from MOLT-4, A, Raji, 293 and MCF-7 cells using #2819. BimEL BimL BimS Events Bim Flow cytometric analysis of Raji cells using #2819 (blue) compared to a nonspecific negative control antibody (red). Bim/Bod is a pro-apoptotic protein belonging to family members containing a BH3 domain but lacking other conserved BH1 or BH2 domains. Bim induces apoptosis by binding to and antagonizing anti-apoptotic members of the family such as, Bcl-xL, Mcl-1, Bcl-w, Bfl-1 and BHRF-1. Three major isoforms of Bim are generated by alternative splicing: BimEL, BimL and BimS. The shortest form, BimS, is the most cytotoxic and is only transiently expressed during apoptosis. BimEL and BimL may be sequestered to the dynein motor complex and released from this complex during apoptosis. Apoptotic activity of these longer isoforms may be regulated by phosphorylation. Environmental stress triggers Bim phosphorylation by JNK, resulting in dissociation with the dynein complex and increased apoptotic activity.

8 Inhibitor of Apoptosis Protein (IAP) Family and Mitochondrial Proteins The inhibitor of apoptosis protein (IAP) family consists of an evolutionarily conserved group of apoptosis inhibitors. Human members of the family include c-iap1, c-iap2, XIAP, survivin, livin and NAIP. Overexpression of IAP family members, particularly survivin and livin, in cancer cell lines and primary tumors suggests a role for these proteins in cancer progression. In general, the IAP proteins function through direct interactions to inhibit caspase activity. In addition, binding of IAP family members to the mitochondrial protein Smac/Diablo blocks its interaction with caspase-9, thereby allowing the processing and activation of the caspase. Mitochondria play a well-established role in the induction of apoptosis. Several proteins such as Smac/Diablo, AIF, HtrA2, endog and cytochrome c are released from mitochondia to promote apoptosis by activating caspases and nucleases or inactivating cytosolic inhibitor proteins. The release of these proteins is regulated by family proteins that control mitochondrial permeability. The anti-apoptotic proteins and Bcl-xL reside in the outer mitochondrial wall and inhibit cytochrome c release. The pro-apoptotic proteins Bad, Bid, Bax and Bim translocate to mitochondria following death signaling, where they promote the release of cytochrome c. The family of proteins is composed of pro- and anti-apoptotic members that homo and heterodimerize with one another. Together, these serve as a rheostat mechanism to regulate the onset of apoptosis at the mitochondrion. In this image, the balance is in favor of the pro-apoptotic Bad proteins (red) that outnumber the anti-apoptotic Bcl-xL proteins (green). As a result, the mitochondrial pore composed of VDAC in the inner membrane and ANT in the outer membrane (both in brown) is shown releasing small molecules into the cytosol during the permeability transition. Cytochrome c (blue) below the outer mitochondrial membrane is poised to spill into the cytosol and trigger apoptosis once the outer mitochondrial membrane ruptures. Applications and Reactivity Key, page 2.

9 Apoptosis Inhibitor Family Survivin (71G4) Rabbit mab #28 Confocal IF analysis of HeLa cells using #28 (green). Mitochondria have been labeled with MitoTracker Red CMXRos (red). Blue = DRAQ5 fluorescent DNA dye. 10 Raji BaF3 C6 Survivin WB analysis of extracts from Raji (human), BaF3 (mouse) and C6 (rat) cell lines using #28. Applications Reactivity c-iap1 Antibody #4952 W H, M, R New c-iap2 (58C7) Rabbit mab #31 W, IP H, (Mk) Events Survivin (71G4) Rabbit mab #28 W, IP, IHC-P, IF-P, F H, M, R Survivin (71G4) Rabbit mab (Alexa Fluor 488 Conjugate) #2810 IF-IC, F H, M, R Survivin (6E4) Mouse mab #22 W H, Mk Survivin Blocking Peptide #1037 XIAP Antibody #42 W, IP, F H, M, Mk XIAP (3B6) Rabbit mab #45 W, IP, IF-IC, F H, Mk Flow cytometric analysis of Jurkat cells using #28 (blue) compared to a nonspecific negative control antibody (red). IHC analysis of paraffin-embedded human transitional epithelial carcinoma of the bladder (left) and human lung carcinoma (right) using #28. Survivin is a 16 anti-apoptotic protein highly expressed during fetal development and cancer cell malignancy. By binding and inhibiting caspase-3, survivin controls the checkpoint in the G2/M-phase of the cell cycle by inhibiting apoptosis and promoting cell division. This regulatory process requires the phosphorylation of survivin at Thr34 by p34 cdc2 kinase. Gene targeting using a Thr34 phosphorylation-defective survivin mutant, as well as antisense survivin, has been shown to inhibit tumor growth. events Survivin IHC analysis of frozen H16 xenograft, showing nuclear localization, using #28. Survivin (71G4) Rabbit mab (Alexa Fluor 488 Conjugate) #2810 Confocal IF analysis of HeLa cells using #2810 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye. Survivin alexa Fluor 488 Flow cytometric analysis of untreated (green) and etoposide-treated (blue) Jurkat cells, using #2810, showing a decrease in survivin expression in apoptotic cells. Akt Signaling Akt/PKB plays a critical role in controlling several cellular processes including survival and apoptosis. Insulin and various growth and survival factors activate Akt through a wortmannin-sensitive pathway involving PI3 kinase. Akt is activated by phospholipid binding and activation loop phosphorylation at Thr8 by PDK1 and by carboxyterminal phosphorylation at Ser473 by TORC2, a protein complex consisting of mtor, rictor and SIN1. Akt promotes cell survival by phosphorylating and inactivating pro-apoptotic targets, including Bad, forkhead transcription factors, c-raf and caspase-9. PTEN phosphatase is a major negative regulator of the PI3 kinase/akt signaling pathway. Akt also promotes cell growth by activating mtor signaling. Akt directly phosphorylates mtor in the mtor-raptor complex (TORC1). Akt also phosphorylates and inactivates TSC2, an inhibitor of TORC1. Moreover, the Akt substrate PRAS binds raptor to inhibit TORC1, while phosphorylation of PRAS by Akt alleviates this interaction. Phospho-Akt (Ser473) (D9E) Rabbit mab # Events Phospho-Akt (Ser473) Flow cytometric analysis of Jurkat cells, untreated (green) or treated with LY2902, wortmannin and U0126 (blue), using # compared to a nonspecific negative control antibody (red). 100 PC3 + + NIH/3T3 + Phospho-Akt (Ser473) Akt Wortmanin LY2902 PDGF WB analysis of extracts from PC3 cells, untreated or LY2902/wortmannintreated, and NIH/3T3 cells, serumstarved or PDGF-treated, using # (upper) or Akt Antibody #9272 (lower). Confocal IF analysis of C2C12 cells, LY2902-treated (left) or insulin-treated (right), using # (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye. For a complete listing of our products see

10 Mitochondrial Proteins Applications Reactivity AIF Antibody #4642 W, IP, IHC-P, IF-IC H, M, R New Bit1 Antibody #3543 W H COX IV Antibody #4844 W, IP, IHC-P, IF-IC, F H, M, R, Mk COX IV (3E11) Rabbit mab #48 W, IP, IHC-P IHC-F, IF-IC, F H, R, Mk New COX IV (3E11) Rabbit mab (Alexa Fluor 488 Conjugate) #48 IF-F, IF-IC, F H, R, Mk Cytochrome c Antibody #4272 W, IHC-P H, M, R, Mk, (B) Cytochrome c (136F3) Rabbit mab #42 W, IHC-P H, M, R, Mk Cytochrome c Blocking Peptide #1033 New Endonuclease G Antibody #4969 W H, M, R, (Mk) HtrA2 Antibody #2176 W H, M, R, Mk Smac/Diablo Mouse mab #2954 W, IP, IHC-P, IF-IC H, Mk SOD1 Antibody #2770 W, IF-IC, F H Thioredoxin 1 Antibody (Human Specific) #2285 W H Thioredoxin 1 Antibody (Mouse/Rat Specific) #2298 W M, R Thioredoxin 1 (C63C6) Rabbit mab #2429 W, IHC-P H, M, R VDAC Antibody #4866 W, IHC-P, IF-IC, F H, M, R AIF (apoptosis-inducing factor) is a ubiquitous flavoprotein critical to caspase-independent apoptosis that is localized to the mitochondrial intermembrane space and released in response to apoptotic stimuli. Treatment of isolated nuclei with recombinant AIF leads to early apoptotic events such as chromatin condensation and large-scale DNA fragmentation. Studies of AIF (-/-) mice have shown that AIF is required for embryoid body cavitation, which results from an early wave of apoptosis during embryonic morphogenesis. Structural analysis revealed two important regions within AIF, the first being a potential DNA binding domain and the second having oxidoreductase activity. Recent studies suggest two functions for AIF: pro-apoptotic via DNA binding and anti-apoptotic via radical scavenging through its oxidoreductase activity. AIF Antibody #4642 AIF Mitochondria Merge 100 NIH/ Jurkat 3T3 NBT-II AIF WB analysis of extracts from human (Jurkat), mouse (NIH/3T3), and rat (NBT-II) cell lines, using #4642. IHC analysis of paraffin-embedded human prostate carcinoma, showing perinuclear and cytoplasmic localization, using #4642. Confocal IF analysis of HeLa cells, using #4642 (green) showing colocalization with mitochondria that have been labeled with MitoTracker Red CMXRos (red). Blue = DRAQ5 fluorescent DNA dye. Smac/Diablo Mouse mab # HeLa Jurkat Smac/Diablo WB analysis of extracts from 293, HeLa and Jurkat cells using #2954. Confocal IF analysis of MCF-7 cells using #2954 (green, top) or with MitoTracker Red CMXRos (red, bottom), which stains mitochondria, demonstrating colocalization. Blue = DRAQ5 fluorescent DNA dye. Smac/Diablo is a 21 mammalian mitochondrial protein that functions as a regulatory component during apoptosis. Upon mitochondrial stress, Smac/Diablo is released from mitochondria and competes with caspases for binding of IAPs (inhibitor of apoptosis proteins). The interaction of Smac/Diablo with IAPs relieves the inhibitory effect of the IAPs on caspases. This interaction involves mainly the amino-terminal residues of Smac/Diablo with the BIR3 region of XIAP, supplemented with several other hydrophobic interactions between the helical structures of Smac/Diablo and other areas of BIR3. Cytochrome c (136F3) Rabbit mab #42 10 HeLa Jurkat COS CHO WB analysis of cell lysates from various cell types using #42. Cytochrome c IHC analysis of paraffin-embedded human lung carcinoma using #42 in the presence of control peptide (top) or Cytochrome c Blocking Peptide #1033 (bottom). Cytochrome c is a well conserved electron-transport protein that is part of the respiratory chain localized to mitochondrial intermembrane space. Upon apoptotic stimulation, cytochrome c released from mitochondria associates with procaspase-9 (47 )/Apaf 1. This complex processes caspase-9 from an inactive proenzyme to its active form. This event further triggers caspase-3 activation and eventually leads to apoptosis. 10 Applications and Reactivity Key, page 2.

11 TNFR Family and Adaptor Proteins The tumor necrosis factor receptor family, which includes TNF- RI, Fas, DR3, DR4, DR5 and DR6, plays an important role in the regulation of apoptosis. The receptors are activated by a family of cytokines that include TNF, FasL and TRAIL. They are characterized by a cysteine-rich extracellular region and an intracellular death domain (DD). The DD recruits other DD containing adaptor proteins (FADD, TRADD, RIP) to the death-inducing signaling complex (DISC) resulting in activation of caspases. Adaptor Proteins Applications Reactivity Apaf-1 Antibody #4452 W H, M, R, Mk Aven Antibody # W, IF-IC, F H, M, R, Mk CRADD/RAIDD Antibody #4899 W H, M, R Phospho-FADD (Ser194) Antibody (Human Specific) #2781 W H Phospho-FADD (Ser191) Antibody (Mouse Specific) #2785 W M FADD Antibody (Human Specific) #2782 W H FAF1 Antibody #4932 W H, M, R FLIP Antibody #3210 W, IF-IC H, M, R NALP1 Antibody #4990 W H, R, (Mk) Nod1 Antibody #3545 W H, M, R, Mk Phospho-PEA-15 (Ser104) Antibody #2776 W, IP H, R, (M) New PEA-15 Antibody #27 W, IP H, M, R, Mk RIP Antibody #4926 W, IP, IF-IC, F H, M, R RIP2 Antibody #4982 W H, M, Mk, (R) RIP2 Kinase #7708 TANK Antibody #2141 W, IP H, M, R, (Mk, B) TRADD (7G8) Rabbit mab #3684 W, IP H TRAF1 (45D3) Rabbit mab #4715 W, IP, IHC-P, IF-IC, F H, (Mk) TRAF1 (1F3) Rat mab #4710 W, IP, IHC-P, IF-IC H, M, R TRAF2 Antibody #4712 W H, M, R, Mk TRAF2 Antibody (Human Specific) #4724 W, IP, IF-IC H, Mk TRAF3 Antibody #4729 W H, M, R, Mk TNFR Family Applications Reactivity DcR1 Antibody #4756 W H, M, R DcR3 Antibody #4758 W H, M, R DR3 Antibody #3254 W H DR5 Antibody #3696 W H FasL Antibody #4273 W, IP, E H RANK Antibody #4845 W H, M, R New TNF-R1 (C25C1) Rabbit mab #3736 W, IP H, M TNF-R2 Antibody #3727 W, IP H, M, R, (Mk) TNF-a Antibody #3707 W, IP H, M, R, Mk TWEAK Receptor/Fn14 Antibody #43 W, IP H, M, R TRAF1 (45D3) Rabbit mab #4715 IHC analysis of paraffin-embedded human tonsil using #4715 preincubated with a control peptide (left) or antigen-specific peptide (right). 100 Raji SR TRAF1 WB analysis of extracts from Raji and SR cells using #4715. TRAFs (TNF receptor-associated factors) are adaptors that bind surface receptors and recruit additional proteins to form multiprotein signaling complexes. TRAFs share a carboxy-terminal TRAF domain, which mediates interactions with associated proteins, and many contain amino-terminal Zinc/RING finger motifs. TRAFs 1-6 act as adaptors for receptors involved in regulation of cell survival, proliferation, differentiation and stress response. Upon binding of the extracellular TRAIL ligand (blue), the DR5 death receptor (beige - here used as a structural stand-in for the Fas receptor) trimerizes and serves as a scaffold for intracellular signaling intermediates. FADD (yellow) is initially recruited to the trimerized receptor via its Death Domain, followed by pro-caspase-8 (red) via its Death Effector Domains. When brought into proximity as a result of binding FADD, pro-caspase-8 becomes cleaved, activated, and released into the cytosol where it will, in turn, cleave and activate caspase-3 and lead to apoptosis. For a complete listing of our products see 11

12 Autophagy Autophagy is a catabolic process that results in the autophagosomic-lysosomal degradation of bulk cytoplasmic contents. Autophagy is generally activated by conditions of nutrient deprivation but has also been associated physiological processes such as development, differentiation, neurodegenerative diseases, infection and cancer. The kinase mtor is a critical regulator of autophagy induction. Upon growth-factor mediated activation of PI3K/Akt and MAP kinase signaling, activated mtor promotes cell growth and protein translation, and suppresses autophagy; mtor is negatively regulated by AMPK and p53 signaling pathways, which promote autophagy. The molecular machinery responsible for autophagy was largely discovered in yeast and are referred to as autophagy-related (Atg) genes. Autophagy is induced by a class III PI3 kinase lipid-kinase complex that includes the tumor suppressor Beclin -1 (Atg 6) as an essential component. Two widely conserved ubiquitin-like conjugation systems, LC3 (Atg8)-phosphatdylethanolamine and Atg12-Atg5 function in autophagosomal vesicle formation. Autophagy and apoptosis are connected both positively and negatively. During nutrient deficiency, autophagy functions as a pro-survival mechanism; however, excessive autophagy leads to autophagic cell death, a process morphologically distinct from apoptosis. Despite this morphological difference, extensive cross-talk occurs between autophagy and apoptosis. Several pro-apoptotic signals, such as TNF, TRAIL and FADD, also induce autophagy and pro-survival signaling through the PI3K/Akt/mTOR pathway suppresses autophagy. Additionally, binds Beclin-1 to inhibit Beclin-1-dependent autophagy, thereby functioning both as a pro-survival and as an anti-autophagic regulator. Precursor LC3B (dark red) is cleaved by Atg4 (yellow), then further processed by Atg7 and Atg3 (not shown) to produce a lipidated form (bright red) that becomes associated with autophagosomal membranes (background). Free Atg12 (orange) is conjugated to Atg5 (brown) by a ubiquitin-like interaction; Atg12+5 then bind Atg16 (yellow-brown). This complex is involved in formation of the autophagosome membrane, possibly acting as a scaffold for membrane extension. 12 Applications and Reactivity Key, page 2.

13 Light chain 3 (LC3), which can serve as a marker for autophagy, was originally identified as a subunit of microtubule-associated proteins 1A and 1B (termed MAP1LC3) and was subsequently found to be a homolog of the to the yeast protein Atg8 critical for autophagy. Three human isoforms of LC3 - LC3A, LC3B and LC3C - undergo post-translational modifications during autophagy. LC3 is first cleaved at the carboxy terminus immediately following synthesis to yield a cytosolic form LC3-I. During autophagy LC3-I is converted to LC3-II through lipidation by a ubiquitin-like system involving Atg7 and Atg3 which results in the association of LC3-II with autophagic vesicles. The presence of LC3 in autophagosomes as well as the conversion of LC3 to the lower migrating form LC3-II are used as indicators of autophagy. LC3B Antibody #2775 Apoptosis AMPK Signaling Amino Acids VPS15 PI3K III Beclin1 PI3K/Akt Signaling Autophagy Induction G L Ser2481 Ser2448 P P Raptor Atg1 P Membrane Nucleation mtor PRAS P MAPK/Erk1/2 Signaling p53/genotoxic Stress Sequestration Confocal IF analysis of HeLa cells, untreated (left) or chloroquine-treated (right), using #2775 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye. Phagophore Atg12 Atg5 Atg16 Atg10 Atg7 Atg7 Atg4 Atg3 LC3-I LC3-II Autophagosome Fusion Lysosome Autophagolysosome Applications Reactivity Beclin-1 Antibody #3738 W, IP, IF-IC H, M, R New LC3B Antibody #2775 W, IF-IC H, M, R, (Mk, C, B, X) New Atg5 Antibody #26 W, IP H, (Mk) New Atg7 Antibody #2631 W H, M, R, (Mk) New Atg12 Antibody (Human Specific) #10 W, IP, IF-IC H New Atg12 Antibody (Mouse Specific) #11 W, IP, IF-IC M New PI3K Class III Antibody #3811 W, IP H, M, R Formation of the autophagosome involves a ubiquitin-like conjugation system in which Atg12 is covalently bound to Atg5 and targeted to autophagic vesicles. This conjugation reaction is mediated by the ubiquitin-e1-like enzyme Atg7 and the E2-like enzyme Atg10. Atg12 Antibody (Mouse Specific) #11 Atg12 Antibody (Human Specific) # Confocal IF analysis of HCT-116 cells, untreated (left) or chloroquine-treated (right), using #10 (green). Blue = DRAQ5 fluorescent DNA dye. HCT-116 THP-1 SR PANC-1 Atg12-Atg5 WB analysis of extracts from various cell lines using #10. free Atg12 Confocal IF analysis of NIH/3T3 cells, untreated (left) or chloroquine-treated (right), using #11 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye. Autophagy Signaling In mammalian cells, mtor integrates signals from nutrients and growth factors to control regulation of cell growth and proliferation. mtor is activated by PI3K/Akt and MAP kinase signaling as well as amino acid levels and inhibited by AMPK and p53 signaling, all of which integrate the energy status and genotoxic stress levels within the cell. mtor also functions as major sensor in autophagy signaling. Activation of mtor promotes protein synthesis and inhibits autophagy. In yeast, the Atg1 Ser/Thr kinase complex functions downstream of mtor to regulate various steps of autophagosome formation. However, the role of Atg1 in higher organisms has yet to be elucidated. For a complete listing of our antibodies, kits and related reagents for the study of mtor signaling please go to mtor (7C10) Rabbit mab #2983 IHC analysis of paraffin-embedded human breast carcinoma, showing cytoplasmic localization using #2983. For a complete listing of our products see 13

14 Transcriptional Regulators Apoptosis is initiated by a wide variety of extrinsic or intrinsic cell signals that direct a cell suicide program to alter gene transcription. Several families of transcription factors play a role in regulating programmed cell death positively or negatively, either as essential promoters or inhibitors of apoptosis. Phospho-c-Myc (Thr58/Ser62) Antibody #91 Phospho-c-Myc (Thr58/Ser62) TPA (min) WB analysis of extracts from A431 cells, untreated or TPA-treated, using #91. Transcriptional Regulators Applications Reactivity Mad-1 Antibody #4682 W H, M, R Max Antibody #4732 W, IP H, M, R Phospho-c-Myc (Thr58/Ser62) Antibody #91 W, IP, E, F H, M, R, Mk c-myc Antibody #92 W, IP H, M, R N-Myc Antibody #95 W, IP, IF-IC H, M, (R, Mk) Nur77 Antibody #3562 W H, M, R, Mk Phospho-PAR-4 (Thr163) Antibody #2329 W H, (M, R, Mk) PAR-4 Antibody #2328 W, IP, IF-IC H, M, R, Mk Phospho-c-Myc (Thr58/Ser62) DNA (PI) 1023 Flow cytometric analysis of Jurkat cells using #91 versus propidium iodide (DNA content). The boxed population indicates phospho-c-myc-positive cells. Confocal analysis of double immunostaining using #91 (green) and calbindin antibody (red) in rat CA1 neurons. (Provided by Dr. Bingren Hu, University of Miami School of Medicine, Florida.) Cytoplasm Nucleus c,n,l-myc Max E-Box Mad1-4 Max E-Box Mnt Max E-Box Mga Max E-Box Gene Transcription X X X The oncoprotein Myc regulates genes involved in cell cycle regulation, metabolism, protein synthesis and mitochondrial function. Myc also represses genes promoting cell growth arrest and cell adhesion. Conversely, overexpression of c-myc sensitizes cells to apoptosis under certain conditions, indicating that Myc has both proliferative and pro-apoptotic effects. Myc is a member of the Myc/Max/Mad family of basic region/helixloop-helix/leucine zipper transcription factors and forms heterodimers with Max, which then bind to E-box sequences to activate transcription. Mad proteins interfere with Myc by inhibiting Myc-mediated transcription. Phosphorylation at Thr58 and Ser62 within Myc s transactivaton domain is thought to be involved in transcriptional regulation. Growth, Proliferation, Differentiation, Apoptosis Other Signaling Proteins Applications Reactivity New Alix (3A9) Mouse mab #2171 W, IP DAP1 Antibody #2282 W, IP H, M, R, Mk DAP3 Antibody #2172 W H, M, R DAP5 Antibody #2182 W H DAPK1 Kinase #7647 New Daxx (25C12) Rabbit mab #4533 W, IF-IC H, M, R, (Mk, B) DRAK2 (33D7) Rabbit mab #2294 W, IP, IHC-P, F M DRAK2 Antibody #2268 W, IP M Phospho-Pim-1 (Tyr218) Antibody #4721 W M Pim-1 Antibody #4722 W H, M, R New Pim-2 Antibody #4723 W H, M, R Pim-1 Kinase #7572 H, (Mk) Pim-2 Kinase #7575 New WWOX Antibody #45 W H, M, R, (Mk) 14 Applications and Reactivity Key, page 2. Daxx (25C12) Rabbit mab #4533 Confocal IF analysis of HeLa cells using #4533 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red) K562 A PC12 Daxx WB analysis of extracts from K562 (human), A (mouse) and PC12 (rat) cell lines using #4533. Daxx was originally identified as a protein that bound to the cytoplasmic domain of Fas and enhanced Fas-mediated apoptosis. Additional studies revealed that Daxx also accumulates in the nucleus, where it localizes to promyelocytic leukemia oncogenic domains (PODs). Nuclear interactions have since been observed with transcription factors, histones and histoneassociated proteins. In the cytoplasm, Daxx has been shown to interact with proteins involved in cell death regulation. Loss of Daxx in mice leads to embryonic lethality with extensive developmental apoptosis, suggesting some role for Daxx directly or indirectly in suppressing cell death.

15 NF-kB Antibodies Nuclear factor-κb (NF-κB) transcription factors are recognized as critical regulators of apoptosis. NF-κB generally promotes cell survival by inducing anti-apoptotic genes, while its role in apoptosis is cell type-dependent and in some cases NF-κB can sensitize cells to apoptotic stimuli. The NF-κB/Rel family consists of RelA, c-rel, RelB, NF-κB1 (p105/p) and NF-κB2 (p100/p52). p105 and p100 are processed by the proteasome to produce p and p52, respectively. p/p52 and Rel proteins form dimeric complexes, which bind DNA to regulate transcription. In unstimulated cells, NF-κB is sequestered in the cytoplasm by IκB inhibitory proteins. NF-κB-activating agents induce IκB phosphorylation, targeting them for degradation through a ubiquitin-proteasome pathway and releasing NF-κB to enter the nucleus and regulate gene expression. NF-κB p65 (C22B4) Rabbit mab # HeLa PC12 Neuro2A NF- B p65 WB analysis of extracts from HeLa (human), PC12 (rat) and Neuro2A (mouse) cell lines using #4764. IHC analysis of paraffin-embedded human melanoma using #4764. Confocal IF analysis of HeLa cells, untreated (left) or TNF-treated (right), using #4764 (green). Actin filaments have been labeled with Alexa Fluor 555 phalloidin (red). Blue = DRAQ5 fluorescent DNA dye. For a complete listing of our antibodies, kits and related reagents for the study of NF-kB Signaling, visit p53 Tumor Suppressor Proteins Activation of p53 can lead to either DNA repair, cell cycle arrest or apoptosis. While the mechanism by which p53 determines the choice of cell fate is unclear, evidence suggests that the phosphorylation status of p53 may determine promoter selectivity. For example, phosphorylation at Ser15 and Ser promotes p53 binding to promoters of growth arrest and DNA repair genes. Conversely, phosphorylation of Ser46 following severe DNA damage increases the affinity of p53 for promoters of pro-apoptotic genes, such as p53aip1. The dual-specificity tyrosine-phosphorylation-regulated kinase 2 (DYRK2) has been found to directly phosphorylate p53 at Ser46 upon genotoxic stress. Other pro-apoptotic p53 target genes include FasL, Bax,, PIG (p53 induced genes), Puma and Noxa. Phospho-p53 (Ser46) Antibody # Phospho-p53 (Ser46) Etoposide (hr) WB analysis of extracts from MCF-7 cells treated with etoposide for the indicated at times using #2521. p53 (7F5) Rabbit mab #2527 WB analysis of extracts from 293 and COS cells using # COS Phospho-p53 (Ser46) 0 3 Etoposide (hr) IP from MCF-7 cell extracts treated with etoposide under nondenaturing conditions, using #2521, followed by WB analysis using a monoclonal p53 antibody. p53 Apart from transcriptional regulation of apoptotic genes, p53 also plays a transcription-independent, pro-apoptotic role by interacting directly with antiand pro-apoptotic family members, such as Bcl-xL and Bax, at the mitochondria. IHC analysis of paraffin-embedded human breast carcinoma (left) and colon carcinoma (right) using #2527. For a complete listing of our p53 antibodies, kits and related reagents, visit For a complete listing of our products see 15

16 Reference Pathways Apoptosis Overview FLIP Caspase-7 ER Stress [Ca++] Calpain Caspase-12 Caspase-8,-10 Caspase-9 APP TNF, FasL, TRAIL FADD TRADD ub ASK1 NIK NF- B XIAP ub Caspase-3,-6,-7 Rock Cell Shrinkage Membrane Blebbing JNK Bid HtrA2 ub Caspase-2 RAIDD PIDD DNA Damage Smac/ Diablo p53 Apaf-1 Caspase-9 DNA repair Apoptosis SIR2 tbid Bax Cyto c AIF DNA Fragmentation Bak PKC Endo G Bcl-xL Bim Erk1/2 p90rsk Bad FasL etc. Survival Factors: Growth Factors, Cytokines, etc. FoxO1 Cell Cycle Cdc2 GSK-3 PI3K Akt Pathway Description: Apoptosis is a regulated cellular suicide mechanism characterized by nuclear condensation, cell shrinkage, membrane blebbing and DNA fragmentation. Caspases, a family of cysteine proteases, are the central regulators of apoptosis. Initiator caspases (including caspase-2, -8, -9, -10, -11 and -12) are closely coupled to pro-apoptotic signals. Once activated, these caspases cleave and activate downstream effector caspases (including caspase-3, -6 and -7), which in turn execute apoptosis by cleaving cellular proteins following specific Asp residues. Activation of Fas and TNFR by FasL and TNF, respectively, leads to the activation of caspase-8 and -10. DNA damage induces the expression of PIDD which binds to RAIDD and caspase-2 and leads to the activation of caspase-2. Cytochrome c released from damaged mitochondria is coupled to the activation of caspase-9. XIAP inhibits caspase-3, -7 and -9. Mitochondria release multiple pro-apoptotic molecules, such as Smac/Diablo, AIF, HtrA2 and endog, in addition to cytochrome c. Smac/Diablo binds to XIAP which prevents it from inhibiting caspases. Caspase-11 is induced and activated by pathological proinflammatory and pro-apoptotic stimuli and leads to the activation of caspase-1 to promote inflammatory response and apoptosis by directly processing caspase-3. Caspase-12 and caspase-7 are activated under ER stress conditions. Anti-apoptotic ligands including growth factors and cytokines activate Akt and p90rsk. Akt inhibits Bad by direct phosphorylation and prevents the expression of Bim by phosphorylating and inhibiting the Forkhead family of transcriptional factors (FKHR). FKHR promotes apoptosis by upregulating proapoptotic molecules such as FasL and Bim. Selected Reviews: Danial, N.N. and Korsmeyer, S.J. (04) Cell death: critical control points. Cell 116, Degterev, A. et al. (03) A decade of caspases. Oncogene. 22, Yan, N. and Shi, Y. (05) Mechanisms of apoptosis through structural biology. Annu. Rev. Cell Dev. Biol. 21, Inhibition of Apoptosis Caspase-8/10 FLIP TNF- TNFR1 FADD RIP1 TRAF2 NIK NEMO cdc37 IKK hsp90 I B I B NF- B NF- B ciap1/2 Bax PTEN GSK-3 PI3K PIP3 Akt PDK1 p70s6k Bad [camp] PKA Ras Raf Survival Factors: Growth Factors, Cytokines, etc. Erk1/2 PKC p90rsk Jak Stat1 Src Stat3 Pathway Description: Cell survival requires the active inhibition of apoptosis, which is accomplished by inhibiting the expression of pro-apoptotic factors as well as promoting the expression of antiapoptotic factors. The PI3K pathway, activated by many survival factors, leads to the activation of Akt, an important player in survival signaling. Pten negatively regulates the PI3K pathway. Activated Akt inhibits the pro-apoptotic family member Bad, Bax, caspase-9, GSK-3 and FoxO1 by phosphorylation. Many growth factors and cytokines induce anti-apoptotic family members. The Jaks and Src phosphorylate and activate Stat3, which in turn induces the expression of Bcl-xL and. Erk1/2 and PKC activate p90rsk, which activates CREB and induces the expression of Bcl-xL and. These family members protect the integrity of mitochondria, preventing cytochrome c release and the subsequent activation of caspase-9. TNF-α may activate both pro-apoptotic and anti-apoptotic pathways: TNF-α can induce apoptosis by activating caspase-8 and -10, but can also inhibit apoptosis signaling via NF-κB, which induces the expression of anti-apoptotic genes such as. ciap1/2 inhibit TNF-α signaling by binding to TRAF2. FLIP inhibits the activation of caspase-8. ub XIAP Caspase-3,-6,-7 Smac/ Diablo ub FoxO1 Fas Bim Apaf1 Caspase-9 Cyto c CREB Bcl-xL HSP27 HSP90 Selected Reviews: Beere, H.M. (04) "The stress of dying": the role of heat shock proteins in the regulation of apoptosis. J. Cell Sci. 117, Franke, T.F. et al. (03) PI3K/Akt and apoptosis: size matters. Oncogene 22, Pommier, Y. et al. (04) Apoptosis defects and chemotherapy resistance: molecular interaction maps and networks. Oncogene 23, HSP70 Apoptosis Direct Stimulatory Modification Multistep Stimulatory Modification Tentative Stimulatory Modification Transcriptional Stimulation 16 Direct Inhibitory Modification Multistep Inhibitory Modification Tentative Inhibitory Modification Transcriptional Inhibition

17 CST would like to thank Prof. Junying Yuan, Harvard Medical School, Boston, Massachusetts, for reviewing these diagrams. Death Receptor Signaling MKK7 JNK FasL Fas/ CD95 Daxx DAPK ASK1 Caspase-8,-10 RIP FADD Caspase- Independent Cell Death Bid tbid Caspase-6 FADD Cyto c TNF- TNFR-1 TNFR-2 DR3 APO-3 TRADD Apaf-1 FLIPs TRADD RIP ASK1 Caspase-9 TRAF2 RAIDD HtrA2 Smac Lamin A Actin Gas2 Fodrin Rock-1 ICAD Acinus PARP ub ciap NIK IKK I B ub NF- B NF- B XIAP TNF- Caspase-3 CAD TRAF2 FLIPs RIP TRAF2 TRADD APO-3L/ TWEAK TRADD FADD FADD Caspase-8,-10 Caspase-7 DNA Repair APO-2L/ TRAIL DR4/5 Pathway Description: Apoptosis can be induced through the activation of death receptors including Fas, TNFαR, DR3, DR4 and DR5 by their respective ligands. Death receptor ligands characteristically initiate signaling via receptor oligomerization, which in turn results in the recruitment of specialized adaptor proteins and activation of caspase cascades. Binding of FasL induces Fas trimerization, which recruits initiator caspase-8 via the adaptor protein FADD. Caspase-8 then oligomerizes and is activated via autocatalysis. Activated caspase-8 stimulates apoptosis via two parallel cascades: it can directly cleave and activate caspase-3, or alternatively, it can cleave Bid, a pro-apoptotic family protein. Truncated Bid (tbid) translocates to mitochondria, inducing cytochrome c release, which sequentially activates caspase-9 and -3. TNF-α and DR- 3L can deliver pro- or anti-apoptotic signals. TNFαR and DR3 promote apoptosis via the adaptor proteins TRADD/FADD and the activation of caspase-8. Interaction of TNF-α with TNFαR may activate the NF-κB pathway via NIK/IKK. The activation of NF-κB induces the expression of pro-survival genes including and FLIP, the latter can directly inhibit the activation of caspase-8. FasL and TNF-α may also activate JNK via ASK1/ MKK7. Activation of JNK may lead to the inhibition of by phosphorylation. Selected Reviews: Debatin, K.M. and Krammer, P.H. (04) Death receptors in chemotherapy and cancer. Oncogene 23, Wajant, H. (02) The Fas signaling pathway: more than a paradigm. Science 296, Cell Shrinkage Membrane Blebbing DNA Fragmentation Chromatin Condensation Apoptosis Mitochondrial Control of Apoptosis PKA Survival Factors: Growth Factors, Cytokines, etc. PKC Erk1/2 p90rsk Bad Bad Cytosolic Sequestration HSP PI3K Akt Caspase-3 p70 S6K Calcineurin Apaf-1 Caspase-9 ub XIAP FasL Fas/ CD95 FADD Caspase-8,-10 Bid Mcl-1 Bcl-xL tbid Bak Bak tbid Bax Bax Bad Cyto c Mule Bcl-xL HtrA2 Apoptosis Microtubules LC8 Bim Smac/ Diablo Mcl-1 LC8 Puma AIF Bim Mule Endo G Hrk DP5 Bax Bax Noxa Death Stimuli: Survival Factor Withdrawal p53 ATM/ ATR JNK Hrk DP5 ING2 DNA damage Genotoxic Stress SIRT2 JNK Bax CAMKII Caspase-2 RAIDD PIDD [NAD] Pathway Description: The family of proteins regulate apoptosis by controlling mitochondrial permeability. The antiapoptotic proteins and Bcl-xL reside in the outer mitochondrial wall and inhibit cytochrome c release. The proapoptotic proteins Bad, Bid, Bax and Bim may reside in the cytosol but translocate to mitochondria following death signaling, where they promote the release of cytochrome c. Bad translocates to mitochondria and forms a pro-apoptotic complex with Bcl-xL. This translocation is inhibited by survival factors that induce the phosphorylation of Bad, leading to its cytosolic sequestration. Cytosolic Bid is cleaved by caspase-8 following signaling through Fas: its active fragment (tbid) translocates to mitochondria. Bax and Bim translocate to mitochondria in response to death stimuli, including survival factor withdrawal. Activated following DNA damage, p53 induces the transcription of Bax, Noxa and PUMA. Upon release from mitochondria, cytochrome c binds to Apaf-1 and forms an activation complex with caspase-9. Although the mechanism(s) regulating mitochondrial permeability and the release of cytochrome c during apoptosis are not fully understood, Bcl-xL, and Bax may influence the voltage-dependent anion channel (VDAC), which may play a role in regulating cytochrome c release. Mule/ARF- BP1 is a DNA damage activated E3 ubiquitin ligase for p53 and Mcl-1, an anti-apoptotic member of. Selected Reviews: Cory, S. et al. (03) The family: roles in cell survival and oncogenesis. Oncogene 22, Green, D.R. and Kroemer, G. (04) The pathophysiology of mitochondrial cell death. Science 5, Shmueli, A. and Oren, M. (05) Life, death, and ubiquitin: taming the mule. Cell 121, Translocation Separation of Subunits or Cleavage Products Joining of Subunits Kinase Phosphatase Transcription Factor 17

18 Select Application References Caspase Signaling #9661 Cleaved Caspase-3 (Asp175) Antibody Reis, T. and Edgar, B.A. (04) Cell 117, (IF) Saito, A. et al. (04) J. Neurosci. 24, (IF) Goodyear, C.S. et al. (04) J. Immunol. 172, (F) #9664 Caspase-3 (Asp175) (5A1) Rabbit mab Wada-Hiraike, O. et al. (06) Proc. Natl. Acad. Sci. USA 103, (IHC) Martel, V. et al. (06) Oncogene [in press]. (IF-IC) Cai, C. et al. (06) J. Biol. Chem. 281, (W) #9662 Caspase-3 Antibody Chandrasekar, B. et al. (04) J. Biol. Chem. 279, (W) Hara, H. et al. (02) J. Immunol. 168, (W) Yu, L. et al. (02) EMBO J. 21, (W) #9665 Caspase-3 (8G10) Rabbit mab Su, J. et al. (06) J. Virol., (W) Tahmatzopoulos, A. et al. (05) Oncogene 24, (W) Carrero, J.A. et al. (04) J. Immunol. 172, (W) #2225 Caspase-1 Antibody Feng, Q. et al. (05) Cancer Res. 65, (W) #9762 Caspase-6 Antibody Hara, H. et al. (02) J. Immunol. 168, (W) Le, D.A. et al. (02) Proc. Natl. Acad. Sci. USA 99, (W) Schroder, A. et al. (02) J. Immunol. 168, (W) #9491 Cleaved Caspase-7 (Asp198) Antibody Guegan, C. et al. (01) J. Neurosci. 21, (IHC) Han, H. et al. (01) J. Biol. Chem. 276, (W) Wang, J. et al. (01) Proc. Natl. Acad. Sci. USA 98, (IHC) #9492 Caspase-7 Antibody Lademann, U. et al. (03) Mol. Cell. Biol. 23, (W) Erhardt, J.A. et al. (01) Thromb. Res. 103, (W) #9748 Cleaved Caspase-8 (Asp384) (11G10) Mouse mab Cheong, J-W. et al. (03) Clin. Cancer Res. 9, (W) #9746 Caspase-8 (1C12) Mouse mab Jeong, W. et al. (04) J. Biol. Chem. 279, (W) Ballestrero, A. et al. (04) Clin. Cancer Res. 10, (W) Mongini, P.K.A. et al. (03) J. Immunol. 171, (W) #95 Cleaved Caspase-9 (Asp315) Antibody (Human Specific) Cheong, J-W. et al. (03) Clin. Cancer Res. 9, (W) Mitchell, K.O. et al. (00) Cancer Res., (W) #91 Cleaved Caspase-9 (Asp3) Antibody (Human Specific) Decaudin, D. et al. (02) Cancer Res. 62, (IC) Guegan, C. et al. (01) J. Neurosci. 21, (IHC) Thomas, W.D. et al. (00) J. Immunol. 165, (W, F) #97 Cleaved Caspase-9 (Asp353) Antibody (Rat Specific) Guimarães, C.A. et al. (03) J. Biol. Chem. 278, (IC) #99 Cleaved Caspase-9 (Asp353) Antibody (Mouse Specific) Kim, I.Y. et al. (02) Cancer Res. 62, (W) Monick, M.M. et al. (06) J. Immunol. 177, (W) #2226 Phospho-Caspase-9 (Thr125) Antibody Balsara, R.D. et al. (06) J. Biol. Chem. 281, (W, IP) #92 Caspase-9 Antibody (Human Specific) Chandrasekar, B. et al. (04) J. Biol. Chem. 279, (W) Yu, W. et al. (03) Cancer Res. 63, (W) Bhakar, A.L. et al. (03) J. Neurosci. 23, (W) #94 Caspase-9 Antibody (Mouse Specific) Katoh, I. et al. (04) J. Biol. Chem. 279, (W) Ekert, P.G. et al. (04) J. Cell Biol. 165, (W) Denecker, G. et al. (01) J. Biol. Chem. 276, (W) #96 Caspase-9 Antibody (Rat Specific) Deming, P.B. et al. (04) Mol. Cell Biol. 24, (W) Penchalaneni, J. et al. (04) Biol. Reprod. 71, (W) Marques, C.A. et al. (03) J. Biol. Chem. 278, (W) #9752 Capase-10 Antibody Penchalaneni, J. et al. (04) Biol. Reprod. 71, (W) #22 Caspase-12 Antibody Li, J. et al. (06) J. Biol. Chem. 281, (W) Sanvicens, N. et al. (04) J. Biol. Chem. 279, (W) Tsai, Y.C. et al. (03) J. Biol. Chem. 278, (W) Cleaved Substrates #9541 Cleaved PARP (Asp214) Antibody (Human Specific) Bhakar, A.L. et al. (03) J. Neurosci. 23, (W) Jiang, C. et al. (01) Cancer Res. 61, (W) Brunet, A. et al. (01) Mol. Cell. Biol. 21, (W) #9546 Cleaved PARP (Asp214) (19F4) Mouse mab (Human Specific) Shukla, S. et al. (05) FASEB J. 19, (IHC) Reboredo, M. et al. (04) J. Gen. Virol. 85, (W) #9548 Cleaved PARP (Asp214) (7C9) Mouse mab (Mouse Specific) Nishigaki, K. et al. (03) J. Biol. Chem. 278, (W) #9545 Cleaved PARP (Asp214) Antibody (Rat Specific) Han, H. et al. (01) J. Biol. Chem. 276, (W) Au-Yeung, K.W. et al. (01) Bio. Pharmacol. 62, (W) Erhardt, J.A. et al. (01) Thromb. Res. 103, (W) #9542 PARP Antibody Chandrasekar, B. et al. (04) J. Biol. Chem. 279, (W) Kumar Sinha, C. et al. (03) Cancer Res. 63, (W) Li, J. et al. (02) J. Biol. Chem. 277, (W) Your Total Antibody Company... Did you know that Cell Signaling Technology (CST): :: is the highest quality provider of activationstate-specific and total protein antibodies. :: produces and validates all antibodies in-house in applications including Western blotting, immunoprecipitation, immunofluorescence, immunohistochemistry and flow cytometry. :: has the same scientists who produce the products (and know them best) provide technical support to customers. :: is committed to raising awareness of sustainable business practices and the conservation of the Earth s biodiversity. Please see the CST website, 07/08 catalog or 07 calendar for a description of some of the initiatives that we support. 18

19 #9732 DFF45/DFF35 Antibody Ishitsuka, K. et al. (05) Oncogene 24, (W) Jendrossek, V. et al. (03) Oncogene 22, (W) #2121 Cleaved a-fodrin (Asp1185) Antibody Hagihara, H. et al. (05) Am. J. Physiol. Heart Circ. Physiol. 288, (IHC) Yoshikawa, Y. et al. (05) Am. J. Physiol. Heart Circ. Physiol. 288, (IHC) Wang, J. et al. (04) Cancer Res. 64, (IHC) #31 Cleaved Lamin A (Asp2) Antibody Holubec, H. et al. (05) J. Histochem. Cytochem. 53, (IHC-P) Yamanaka, K. et al. (05) Mol. Cancer Ther. 4, (W) Martin, D.N. and Baehrecke, E.H. (04) Development. 131, (IC-IF) #35 Cleaved Lamin A (Small Subunit) Antibody Martensson, K. et al. (04) J. Bone Miner. Res. 19, (IHC) #32 Lamin A/C Antibody Dentin, R. et al. (04) J. Biol. Chem. 279, (W) Charniot, J.C. et al. (03) Hum. Mutat. 21, (IC-IF) Sun, S. et al. (02) Cancer Res. 62, (W) Kim, K. et al. (02) Mol. Cancer Ther. 1, (W) Family Members #2875 Phospho- (Thr56) Antibody (Human Specific) Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W, IHC) #2876 Antibody Yang, Y.M. et al. (05) Cancer Res. 65, (W) Yang, L. et al. (04) J. Biol. Chem. 279, (W) #2872 Antibody (Human Specific) Samanta, A.K. et al. (04) J. Biol. Chem. 279, (W) #4572 Mcl-1 Antibody Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W) #2762 Bcl-xL Antibody Leu, J. et al. (04) Nat. Cell Biol. 6, (W, IP) Saito, A. et al. (03) J. Neurosci. 23, (W, IHC) Yang, C-C. et al. (03) J. Biol. Chem. 278, (W) #2772 Bax Antibody Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W, IHC) Yang, L. et al. (04) J. Biol. Chem. 279, (W) Rusiñol, A.E. et al. (04) J. Biol. Chem. 279, (W) Leu, J. et al. (04) Nat. Cell Biol. 6, (W, IP) Cheong, J-W. et al. (03) Clin. Cancer Res. 9, (W) #9291 Phospho-Bad (Ser112) Antibody Stein, T.D. et al. (04) J. Neurosci. 24, (IHC) Stein, T.D. and Johnson, J.A. (02) J. Neurosci. 22, (IHC-P) Jamieson, C.A. and Yamamoto, K.R. (00) Proc. Natl. Acad. Sci. USA 97, (W) Bertolotto, C. et al. (00) J. Biol. Chem. 275, (W) Tan, Y. et al. (1999) J. Biol. Chem. 274, (W) #9296 Phospho-Bad (Ser112) (7E11) Mouse mab Avota, E. et al. (01) Nat. Med. 7, (W) Bieberich, E. et al. (01) J. Biol. Chem. 276, (W) #9295 Phospho-Bad (Ser136) Antibody Rusiñol, A.E. et al. (04) J. Biol. Chem. 279, (W) Yang, C C. et al. (03) J. Biol. Chem. 278, (W) #9297 Phospho-Bad (Ser155) Antibody Saito, A. et al. (03) J. Neurosci. 23, (W) Yusta, B. et al. (02) J. Biol. Chem. 277, (W) Tan, Y. et al. (00) J. Biol. Chem. 275, (W) #9292 Bad Antibody Liu, C. et al. (03) Cancer Res. 63, (W, IP) Saito, A. et al. (03) J. Neurosci. 23, (W) Yu, C. et al. (03) Cancer Res. 63, (W) #4976 Puma Antibody Nyman, U. et al. (05) J. Biol. Chem. 2, (IC-IF) #4692 Bmf Antibody Niizuma, H. et al. (06) Oncogene 25, (W) #02 BID Antibody (Human Specific) Abdelrahim, M. et al. (06) Carcinogenesis 27, (W) Weng, C. et al. (05) J. Biol. Chem. 2, (W) Chandrasekar, B. et al. (04) J. Biol. Chem. 279, (W) Apoptosis Inhibitor Family #42 XIAP Antibody Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W, IHC) Rosato, R.R. et al. (03) Mol. Cancer Ther. 2, (W) Guegan, C. et al. (01) J. Neurosci. 2, (W) #22 Survivin (6E4) Mouse mab Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W, IHC) Fukuda, S. et al. (02) Blood 100, (F) Mitochondrial Proteins #2954 Smac/Diablo Mouse mab Gulmann, C. et al. (05) Clin. Cancer Res. 11, (W, IHC) Adaptor Proteins #2781 Phospho-FADD (Ser194) Antibody (Human Specific) Chen, G. et al. (05) Proc. Natl. Acad. Sci. USA 102, (W) Shimada, K. et al. (04) Carcinogenesis 25, (W) #2782 FADD Antibody (Human Specific) Kim, S.H. et al. (04) J. Biol. Chem. 279, (W, IP) #3210 FLIP Antibody Jin, Z. et al. (04) J. Biol. Chem. 279, (W) #4982 RIP2 Antibody Rosenstiel, P. et al. (06) Proc. Natl. Acad. Sci. USA 103, (W) Antibodies, Kits and Kinases Screening Tools and Technologies Phospho-Ser/Thr/Tyr Motif Antibodies Chromatin Regulation MAP Kinase Signaling Apoptosis/Caspase Signaling Akt Signaling Pathways Translational Control Calcium, camp and Lipid Signaling Cell Cycle/Checkpoint Control/DNA Damage Immunology/Inflammation Neuroscience Tyrosine Kinases Adaptor Proteins Cytoskeletal Regulation Glucose/Energy Metabolism Nuclear Receptors and Phosphatases Protein Folding and Stability Development and Differentiation Ancillary Reagents For a complete listing of our products see 19

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