CELLULAR SIGNALING IN NEUROSCIENCE

Transcription

1 Neuro_Broch all pgs 11/3/05 10:16 AM Page 1 CELLULAR SIGNALING IN NEUROSCIENCE General Neuronal Signaling Alzheimer s, Parkinson s and other Neurological Disorders Neuronal Injury and Regeneration

2 Neuro_Broch all pgs 11/3/05 10:16 AM Page 2 INNOVATIVE DISCOVERY TOOLS FOR SIGNAL TRANSDUCTION RESEARCH Table of Contents General Neuronal Signaling pages 3-10 Neuronal Disease: Alzheimer s and Parkinson s Disease pages Neuronal Injury and Regeneration pages Cover Image: Phospho-µ-Opioid Receptor (Ser375) Antibody #3451 Phospho-µ-Opioid Receptor (Ser475) Antibody #3451 (red) showing bright staining in Purkinje cells and other neuronal processes in mouse cerebellum. Green = CREB (86B10) Mouse mab #9104, blue pseudocolor = DRAQ5 fluorescent DNA dye. For more information about our antibodies, please visit our website at 2

3 Neuro_Broch all pgs 11/3/05 10:16 AM Page 3 General Neuronal Signaling A primary function of cellular signaling in neurons, as in most cell types, is to translate information from the outside of the cell to a cellular response inside the cell. In neurons this process may take the form of membrane receptors and neurotransmitters. Synthesis of transmitters, activation of receptors and signaling downstream of the activated receptors are controlled by kinases and other regulators of secondary modification and protein processing. As with other membrane receptors, downstream signaling is also regulated by adaptors and transcription factors. Many of the intermediates are unique to neurons or other neuronal cell types. As with all cell types, neuronal cell death or deregulated proliferation results from a failure in these cellular signaling processes. Specific antibodies that monitor protein modifications and expression are the foundation for studying these processes. The unique properties of neuronal cells and model systems present challenges to the development and use of these antibodies. Dedicated antibody testing and validation using a variety of platforms such as the confocal microscope, frozen sections and appropriate cell and animal models provide the means to meet these challenges. CREB (48H2) Rabbit mab #9197 Confocal immunofluorescent image of GFAP (2A5) Mouse mab #3665 (green) and CREB (48H2) Rabbit mab #9197 (red) staining the CA1 region of rat hippocampus. 3

4 Neuro_Broch all pgs 11/3/05 10:16 AM Page 4 General Neuronal Signaling Phospho-MAP2 (Ser136) Antibody #4541 Phospho-MAP2 (Ser136) Antibody #4541 (red) and CREB (86B10) Mouse mab #9104 (green) staining mouse cerebellum. MAP2 Antibody #4542 MAP2 Antibody #4542 (red) and GFAP (2A5) Mouse mab #3665 (green) staining rat hippocampus. Phospho-MAP2 (Thr1620/1623) Antibody #4544 Phospho-MAP2 (Thr1620/1623) Antibody #4544 (red) and CREB (86B10) Mouse mab #9104 (green) staining mouse hippocampus. 4

5 Neuro_Broch all pgs 11/3/05 10:16 AM Page 5 Phospho-NMDAR1 (Ser890) Antibody #3381 Phospho-NMDAR1 (Ser890) Antibody #3381 (red) staining the outer plexiform layer of mouse retina. Green = CREB (86B10) Mouse mab #9104, blue pseudocolor = DRAQ5 fluorescent DNA dye. Synapsin Antibody #2312 A Synapsin Antibody #2312 (red, A) and Phospho- Synapsin (Ser9) Antibody #2311 (red, B) in mouse retina showing prominent staining in the inner plexiform layer. Green = CREB (86B10) Mouse mab #9104, blue pseudocolor = DRAQ5 fluorescent DNA dye. Phospho-Synapsin (Ser9) Antibody #2311 B 5

6 Neuro_Broch all pgs 11/3/05 10:16 AM Page 6 General Neuronal Signaling Phospho-Tyrosine Mouse mab (P-Tyr-100) #9411 Phospho-Tyrosine Mouse mab (P-Tyr-100) #9411 (green) and MAP2 Antibody #4542 (red) staining in mouse retina. Phospho-tyrosine label is brightest in the nerve fiber layer while MAP2 label is primarily in the plexiform layers. Blue pseudocolor = DRAQ5 fluorescent DNA dye. A Tyrosine Hydroxylase Antibody #2792 Tyrosine Hydroxlyase Antibody #2792 (red, A) and Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 (red, B) staining Purkinje cells (arrowheads) in mouse cerebellum. Blue pseudocolor = DRAQ5 fluorescent DNA dye. B Phospho-Tyrosine Hydroxylase (Ser40) Antibody #2791 6

7 Neuro_Broch all pgs 11/3/05 10:16 AM Page 7 Neurofilament-L (DA2) Mouse mab #2835 Neurofilament-L (DA2) Mouse mab #2835 (green) and CREB (48H2) Rabbit mab #9197 (red) staining in mouse cerebellum. Blue pseudocolor = DRAQ5 fluorescent DNA dye. CaMKII Antibody #3362 A CaMKII Antibody #3362 (red, A) and Phospho-CaMKII (Thr286) Antibody #3361 (red, B) staining in mouse retina. Green = CREB (86B10) Mouse mab #9104, blue pseudocolor = DRAQ5 fluorescent DNA dye. Phospho-CaMKII (Thr286) Antibody #3361. B 7

8 Neuro_Broch all pgs 11/3/05 10:16 AM Page 8 General Neuronal Signaling Phospho-δ-Opioid Receptor (Ser363) Antibody #3641 Phospho-δ-Opioid Receptor (Ser363) Antibody #3641 (red) and CREB (86B10) Mouse mab #9104 (green) staining mouse cerebellum. Blue pseudocolor = DRAQ5 fluorescent DNA dye. β-tubulin Antibody #2146 β-tubulin Antibody #2146 showing cytoskeletal staining in SH-SY5Y human neuroblastoma cells. α/β-tubulin Antibody #2148 α/β-tubulin Antibody #2148 showing cytoskeletal staining in SH-SY5Y human neuroblastoma cells. 8

9 Neuro_Broch all pgs 11/3/05 10:17 AM Page 9 DARPP-32 Antibody #2302 A DARPP-32 Antibody #2302 (red, A), Phospho-DARPP-32 (Thr34) Antibody #2304 (red, B), and Phospho- DARPP-32 (Thr75) Antibody (red, C) staining ganglion cells and plexiform layers of mouse retina compared to a non-specific control antibody (red, D). Blue pseudocolor = DRAQ5 fluorescent DNA dye. Phospho-DARPP-32 (Thr34) Antibody #2304 B Phospho-DARPP-32 (Thr75) Antibody #2301 C Non-specific control antibody D 9

10 Neuro_Broch all pgs 11/3/05 10:17 AM Page 10 General neuronal signaling ANTIBODIES PRODUCT # APPLICATIONS REACTIVITY N-Cadherin Antibody 4061 W, IP H, M, R, Mk Calpain 1 Large Subunit (Mu-type) Antibody 2556 W H, M, (R) Calpain 2 Large Subunit (M-type) Antibody 2539 W, IP, F H, M, R Phospho-CaMKII (Thr286) Antibody 3361 IF-F, W H, M, R CaMKII Antibody 3362 IF-F, W H, M, R, Mk CaMKIV Antibody 4032 W, IP H, M, R CD56 (NCAM) (123C3) Mouse mab 3576 IHC-P, W, F H Phospho-CREB (Ser133) (1B6) Mouse mab 9196 W H, M, R Phospho-CREB (Ser133) (87G3) Rabbit mab 9198 IF-IC, IF-F, IHC-P, W, F H, M, R Phospho-CREB (Ser133) Antibody 9191 IF-IC, IF-F, IHC-P, W, IP H, M, R CREB Antibody 9192 IF-IC, IHC-P, W, IP H, M, R, C CREB (48H2) Rabbit mab 9197 IF-IC, IF-F, IHC-P, W, IP H, M, R, Mk CREB (86B10) Mouse mab 9104 IF-IC, IF-F, W, F H, M, R, Mk Phospho-Dab1 (Tyr220) Antibody 3327 W H Phospho-Dab1 (Tyr232) Antibody 3325 W H Dab1 Antibody 3328 W H EGR1 Antibody 4152 IF-IC, W, IP R, (H, M) Phospho-DARPP-32 (Thr34) Antibody 2304 IF-F, W H, M, R Phospho-DARPP-32 (Thr75) Antibody 2301 IF-F, W H, M, R DARPP-32 Antibody 2302 IF-F, IHC-P, W H, M, R Phospho-Erk5 (Thr218/Tyr220) Antibody 3371 W H, M, R Phospho-FLT3 (Tyr591) Antibody 3461 W, IP H, M GFAP (2A5) Mouse mab 3665 W, IF-IC H, M Phospho-MAP2 (Ser136) Antibody 4541 IF-F, W H, M, R, Mk Phospho-MAP2 (Thr1620/1623) Antibody 4544 IF-F, W H, R, (M) MAP2 Antibody 4542 IF-IC, IF-F, W H, R, (M) Merlin (1C4) Mouse mab 9169 IHC-P, W, IP H, M, R Merlin Antibody 9168 IHC-P, W, IP H, M, R MLK3 Antibody 2817 W H, M, R, Hm, Mk Neurofilament-L (DA2) Mouse mab 2835 W, IF-IC H, M, R Phospho-NMDAR1 (Ser890) Antibody 3381 IF-F, W, F H, M, R Phospho-NMDAR1 (Ser896) Antibody 3384 W H, M, R Phospho-NMDAR1 (Ser897) Antibody 3385 W H, M, R Phospho-δ-Opioid Receptor (Ser363) Antibody 3641 IF-IC, IF-F, W H, M Phospho-µ-Opioid Receptor (Ser375) Antibody 3451 IF-IC, IF-F, W, IP M, (H) Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mab 9215 IF-IC, W, IP, F H, M, R, Mk Phospho-p38 MAPK (Thr180/Tyr182)(12F8) Rabbit mab (IHC Preferred) 4631 IF-IC, IHC-P, W, IP H, M, R, Mk Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab 9216 W, IP, F H,M, R Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab (Alexa Fluor 647 Conjugate) 4552 F H, M Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab (Alexa Fluor 488 Conjugate) 4551 F H, M Phospho-p38 MAP Kinase (Thr180/Tyr182) Antibody 9211 IF-IC, W, IP, F H, M, R p38 MAP Kinase (5F11) Mouse mab 9217 W, IP, F H, M, R p38 MAPK (5F11) Mouse mab (Alexa Fluor 647 Conjugate) 4555 F H p38 MAPK (5F11) Mouse mab (Alexa Fluor 488 Conjugate) 4556 F H p38 MAP Kinase (L53F8) Mouse mab 9228 W, IP, F H, M, R, Mk p38 MAP Kinase Antibody 9212 IHC-P, W, F H, M, R p38 MAP Kinase α Antibody 9218 IHC-P, W, IP H, M, R, Mk p38 MAP Kinase δ Antibody 9214 W, IP H, M, R, Mk Parkin Antibody 2132 IF-IC, W, IP H, M, R Phospho-PKC (pan) (βii Ser660) Antibody 9371 IF-IC, W, IP H, M, R, Mk Phospho-PKC (pan) (γ Thr514) Antibody 9379 W, IP H, M, R, Mk Phospho-Ras-GRF1 (Ser916) Antibody 3321 W M, R Ras-GRF1 Antibody 3322 IHC-P, W M Phospho-Synapsin (Ser9) Antibody 2311 IF-F, W M, R, (H) Synapsin Antibody 2312 IF-F, W M, R, (H) α-synuclein Antibody 2642 W, IP H, M, R, Mk α/β-synuclein (Syn205) Mouse mab 2644 IHC-P, W, IP M, R, (H) Phospho-TrkA (Tyr490) Antibody 9141 W, IP R, (H, M) α-tubulin Antibody 2144 IHC-P, W, F H, M, R β-tubulin Antibody 2146 IF-IC, IHC-P, W, F H, M, R Phospho-Tyrosine Hydroxylase (Ser40) Antibody 2791 IF-IC, IF-F, W R, (H, M) Tyrosine Hydroxylase Antibody 2792 IF-IC, IF-F, W H, M, R Phospho-Tyrosine Mouse mab (P-Tyr-100) 9411 IF-F, IHC-P, W, IP, F, E, D All Phospho-Tyrosine Mouse mab (P-Tyr-100) (Alexa Fluor 488 Conjugate) 9414 IF-IC, F H Phospho-Tyrosine Mouse mab (P-Tyr-100) (Alexa Fluor 647 Conjugate) 9415 IF-IC, F H Applications Reactivity IF-IC Immunofluorescence (Immunocytochemistry) IF-F Immunofluorescence (Frozen) IF-P Immunofluorescence (Paraffin) IHC-P Immunohistochemistry (Paraffin) W Western IP Immunoprecipitation F Flow cytometry E ELISA D DELFIA H Human M Mouse R Rat Hm Hamster Mk Monkey C Chicken X Xenopus All All species expected ( ) 100% sequence homology

11 Neuro_Broch all pgs 11/3/05 10:17 AM Page 11 Neuronal Disease: Alzheimer s and Parkinson s Diseases Neurological diseases including Alzheimer s and Parkinson s diseases are best known for accumulations of misprocessed proteins in the diseased brain. The accumulation of Aβ in Alzheimer s Disease and α-synuclein in Parkinson s Disease is known to be associated with, if not causative of these disorders. Related genetic mutations in genes such as APP, α-synuclein, parkin, Apo E and presenilins and alternative splicing of proteins including tau are also known to be associated with diseases involving protein aggregations. All of these processes are regulated by cellular signaling events, some of which have been identified. As many as 25 phosphorylation sites on tau, a protein that is found in protein aggregations, may be regulated by upstream kinases including GSK-3 and Erk. Protein cleavage and secondary modifications are other forms of regulation that are central to these diseases. APP cleavage to Aβ and cleavage of tau by caspases are events associated with Alzheimer s Disease. Misregulation of tau is thought to be interconnected with cleavage of APP through p38 MAP Kinase, GSK-3 and the presenilins. The dominant model for Alzheimer s Disease is the Amyloid Cascade Hypothesis although alternative models have been proposed. In addition, exposure to environmental toxins can result in oxidative stress and nitration of α-synuclein as well as changes in mitogenic signaling that may lead to cellular conditions that predispose individuals to these diseases. Furthermore, research on misprocessed proteins can contribute to our fundamental understanding of cellular signaling, as demonstrated by the discovery of regulated intramembranous proteolysis (RIP), a process which was identified through studies on APP processing. β-amyloid Antibody #2454 β-amyloid Antibody #2454 (red) and Tau (Tau46) Mouse mab #4019 (green) staining in human Alzheimer s brain. Blue pseudocolor = DRAQ5 fluorescent DNA dye. 11

12 Neuro_Broch all pgs 11/3/05 10:17 AM Page 12 Neuronal Disease: Alzheimer s and Parkinson s Diseases Tau (Tau46) Mouse mab #4019 Tau (Tau46) Mouse mab #4019 (green) and APP Antibody #2452 (red) staining in human Alzheimer s brain. APP/β-Amyloid (NAB228) Mouse mab #2450 APP/β-Amyloid (NAB228) Mouse mab #2450 (green) and Phospho-p44/42 MAPK (Thr202/Tyr204) (197G2) Rabbit mab #4377 (red) staining in human Alzheimer s brain. Blue pseudocolor = DRAQ5 fluorescent DNA dye. p35 Antibody #2673 p35 Antibody #2673 (red) staining Purkinje cells in mouse cerebellum. Green = CREB (86B10) Mouse mab #9104, blue pseudocolor = DRAQ5 fluorescent DNA dye. 12

13 Neuro_Broch all pgs 11/3/05 10:17 AM Page 13 APP Antibody #2452 APP Antibody #2452 (red) staining the cytoplasm of SH-SY5Y human neuroblastoma cells. Blue pseudocolor = DRAQ5 fluorescent DNA dye. Presenilin 2 Antibody #2192 A Presenilin 2 Antibody #2192 (red, A) staining PC-12 rat pheochromocytoma cell line compared to an isotype control (B). Blue pseudocolor = DRAQ5 fluorescent DNA dye. Isotype control B 13

14 Neuro_Broch all pgs 11/3/05 10:17 AM Page 14 Neuronal Disease: Alzheimer s and Parkinson s Diseases ANTIBODIES PRODUCT # APPLICATIONS REACTIVITY β-amyloid Antibody 2454 IF-P, W H, (R) Phospho-APP (Thr668) Antibody 2451 IHC-P, W, IP H, M, R APP/β-Amyloid (NAB228) Mouse mab 2450 IF-P, IHC-P, W H, M, (R, Mk) APP Antibody 2452 IF-IC, IF-F, IF-P IHC-P, W H, M, R, Mk Phospho-CaMKII (Thr286) Antibody 3361 IF-F, W H, M, R CaMKII Antibody 3362 IF-F, W H, M, R, Mk Casein Kinase I Antibody 2655 W H, M, (R, C, X) CKII-α Antibody 2656 W H, M Cleaved Caspase-3 (Asp175) (5A1) Rabbit mab 9664 IF-IC, IHC-P, W, IP, F H, M, R Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor 488 Conjugate) 9669 IF-IC, F H Cleaved Caspase-3 (Asp175) Antibody 9661 IF-IC, IHC-P, W, F H, M, R Caspase-3 (3G2 ) Mouse mab 9668 W H Caspase-3 (8G10) Rabbit mab 9665 IF-IC, W, IP H, M, R Caspase-3 Antibody 9662 IF-IC, IHC-P, W, IP H, M, R Phospho-GSK-3β (Ser9) Antibody 9336 IF-IC, IHC-P, W H, M, R, Mk, Z Nicastrin Antibody 3632 W H, M, R, Mk Cleaved Notch 1 (Val1744) Antibody 2421 IF-IC, W, IP, E H, M, R, Mk p35 Antibody 2673 IF-F, W, IP M, R, (H) Phospho-p44/42 MAPK (Thr202/Tyr204) (20G11) Rabbit mab (IHC Preferred) 4376 IF-IC, IHC-P, W H, M, R Phospho-p44/42 MAPK (Thr202/Tyr204) (197G2) Rabbit mab 4377 IF-F, IF-IC, W, F H, M, R Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab 9106 IF-IC, W, IP, F H, M, R, Hm, Z Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab (Alexa Fluor 488 Conjugate) 4374 F H, M Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab (Alexa Fluor 647 Conjugate) 4375 F H, M Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody 9101 IF-IC, W, IP, F H, M, R, C, Hm, Z p44/42 MAP Kinase (137F5) Rabbit mab 4695 IHC-P, W, IP, F H, M, R, Mk p44/42 MAP Kinase (L34F12) Mouse mab 4696 IHC-P, W, IP, F H, M, R, Mk p44/42 MAP Kinase Antibody 9102 IHC-P, W, F H, M, R, Hm, Z Phospho-PKA C (Thr197) Antibody 4781 W, F M, R, (H) PKA C-α Antibody 4782 W, IP, F H, M, R Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mab 9624 W, IP, E, D All Phospho-(Ser/Thr) PKA Substrate Antibody 9621 IHC-P, W, IP, E, D All Presenilin 1 Antibody 3622 W, IP H, R, Mk, (M) Presenilin 2 Antibody 2192 IF-IC, IHC-P, W, IP H, M, R, Mk Tau (Tau46) Mouse mab 4019 IF-F, IF-P, IHC-P, W, IP H, M, R α/β-tubulin Antibody 2148 IF-IC, IHC-P, W, F H, M, R, Mk Harmless Fragments Normal α-secretase γ-secretase Alzheimer s Disease Signaling The Amyloid Cascade Hypothesis Amyloid Precursor Protein (APP) Alzheimer s β-secretase γ-secretase (presenilin 1 & 2) Aβ(1-42) Insoluble Plaques Inflammation Oxidative Stress Ionic Homeostasis Dysregulation Aberrant Kinase Activation Akt GSK-3β Erk2 CaMKII CDK5/p35 CK PKA p38 Tau Hyperphosphorylation Microtubule Destabilization Neurofibrillary Tangles Neuronal Dysfunction/Death Dementia Selkoe DJ (1991) Neuron Apr.6, Verdile et al. ( 2004) Pharmacol Res. 50, Applications Reactivity IF-IC Immunofluorescence (Immunocytochemistry) IF-F Immunofluorescence (Frozen) IF-P Immunofluorescence (Paraffin) IHC-P Immunohistochemistry (Paraffin) W Western IP Immunoprecipitation F Flow cytometry E ELISA D DELFIA H Human M Mouse R Rat Hm Hamster Mk Monkey C Chicken X Xenopus All All species expected ( ) 100% sequence homology

15 Neuro_Broch all pgs 11/3/05 10:17 AM Page 15 Neuronal Injury and Regeneration Cellular responses to neurological injuries that result in ischemia, including stroke, consist primarily of cell death or cell survival and protective mechanisms that may later lead to cell regeneration. Cell death or survival responses originate from the activation of basic signaling pathways such as the p38 MAP Kinase and SAPK/JNK stress response pathways leading to cellular apoptosis or other outcomes. Cell protective mechanisms such as induction of hypoxia inducible factors, heat shock proteins or anti-apoptotic factors such as IAP and Bcl2 family members may also occur. Activation of the Akt survival pathway is associated with ischemia, resulting in regulation of many different cellular functions including protein translation, apoptosis, metabolic regulation, synaptic signaling and cell proliferation. Akt activation, along with signals from other pathways, can induce stem cell activation and neuronal regeneration. The rat brain ischemia and reperfusion model provides researchers with a system for the identification of pathways and proteins that regulate cellular responses to injury as well as regeneration. Activation of stress kinases, cleavage of caspases indicative of apoptosis and activation of Akt and other stem cell proliferation pathways, as demonstrated by histone H3 phosphorylation, may all be studied using this model system. EGR1 Antibody #4152 EGR1 Antibody #4152 (red) and Phospho-S6 Ribosomal Protein (Ser225/236) (2F9) Rabbit mab (Alexa Fluor 488 Conjugate) #4854 (green) staining the subventricular zone of the striatum in reperfused ischemic rat brain. Proliferating cells are labeled with Phospho-Histone H3 (Ser10) (6G3) Mouse mab #9706 (blue). 15

16 Neuro_Broch all pgs 11/3/05 10:17 AM Page 16 Neuronal Injury and Regeneration Phospho-Akt (Ser473) (193H12) Rabbit mab #4058 Phospho-Akt (Ser473) (193H12) Rabbit mab #4058 (red) and Phospho-Histone H3 (Ser10) Antibody (Alexa Fluor 488 Conjugate) #9708 (green) staining the subventricular zone in rat striatum following unilateral ischemia/reperfusion. Ischemia Phospho-CREB (Ser133) (87G3) Rabbit mab #9198 Phospho-CREB (Ser133) (87G3) Rabbit mab #9198 (red) and c-jun (L70B11) Mouse mab #2315 (green) in rat striatum. Ischemia/reperfusion induces CREB phosphorylation and c-jun expression in the subventricular zone. Sham 16 Ischemic rat brain sections kindly provided by Dr Bingren Hu, University of Miami School of Medicine.

17 Neuro_Broch all pgs 11/3/05 10:17 AM Page 17 Phospho-PTEN (Ser380) Antibody #9551 Phospho-PTEN (Ser380) Antibody #9551 staining in rat striatum. Ischemia induces PTEN phosphorylation in the subventricular zone following repurfusion. Sham Ischemia Sham Phospho-PTEN (Ser380) Antibody #9551 Low magnification mosaic of Phospho-PTEN (Ser380) Antibody #9551 staining in rat striatum. Ischemia/reperfusion followed by 30 minutes or 4 hours reperfusion induces PTEN phosphorylation specifically in the subventricular zone (arrowhead). 30 minutes 4 hours 17

18 Neuro_Broch all pgs 11/3/05 10:17 AM Page 18 Neuronal Injury and Regeneration ANTIBODIES PRODUCT# APPLICATIONS REACTIVITY Phospho-Akt Substrate (RXRXXS/T) (110B7) Rabbit mab 9614 IHC-P, W, IP, E, D All Phospho-(Ser/Thr) Akt Substrate Antibody 9611 IHC-P, W, IP, E, D All Phospho-Akt (Ser473) (736E11) Rabbit mab (IHC Specific) 3787 IHC-P H, (M, R) Phospho-Akt (Ser473) (193H12) Rabbit mab 4058 IF-F, W, IP, F H, M, R Phospho-Akt (Ser473) (193H12) Rabbit mab (Alexa Fluor 488 Conjugate) 2336 F H Phospho-Akt (Ser473) (193H12) Rabbit mab (Alexa Fluor 647 Conjugate) 2337 F H Phospho-Akt (Ser473) (587F11) Mouse mab 4051 IHC-P, W, IP H, M, R, Hm Phospho-Akt (Ser473) Antibody 9271 W, IP, F H, M, R, Hm, C Phospho-Akt (Thr308) (244F9) Rabbit mab 4056 IHC-P, W, F H, M, R Phospho-Akt (Thr308) Antibody 9275 W, IP, F H, M, R, Hm, (C, X) Phospho-Akt (Tyr326) Antibody 2968 W M, (H, R) Phospho-Drosophila Akt (Ser505) Antibody 4054 IF-IC, W Dr Akt (5G3) Mouse mab 2966 IF-IC, IP H, M, R, Hm Akt Antibody 9272 W, IP, F H, M, R, Hm, C, Dr Akt1 (2H10) Mouse mab 2967 IHC-P, W, IP, F H, M, R Akt2 (54G8) Rabbit mab (IHC Specific) 4057 IHC H, (M, R) Akt2 (5B5) Rabbit mab 2964 W, IP H, M, R, Mk Akt2 Antibody 2962 W, IP H, M, R Akt3 (62A8) Rabbit mab 3788 IHC-P, W, IP H, M, R Akt3 Antibody 4059 IHC-P, W, IP H, M, R Phospho-Bad (Ser112) (7E11) Mouse mab 9296 IHC-P, W, IP H, M, R, Mk Phospho-Bad (Ser112) Antibody 9291 W, IP, F H, M, R, Mk Bad Antibody 9292 IF-IC, IHC-P, W, IP H, M, R, Mk Phospho-Bcl-2 (Thr56) Antibody (Human Specific) 2875 W H Phospho-Bcl-2 (Ser70) Antibody (Human Specific) 2871 W, F H Bcl-2 Antibody 2876 W H, M, R Bcl-2 Antibody (Human Specific) 2872 W, IP H Cleaved Caspase-3 (Asp175) (5A1) Rabbit mab 9664 IF-IC, IHC-P, W, IP, F H, M, R Cleaved Caspase-3 (Asp175) Antibody (Alexa Fluor 488 Conjugate) 9669 IF-IC, F H Cleaved Caspase-3 (Asp175) Antibody 9661 IF-IC, IHC-P, W, F H, M, R Caspase-3 (3G2 ) Mouse mab 9668 W H Caspase-3 (8G10) Rabbit mab 9665 IF-IC, W, IP H, M, R Caspase-3 Antibody 9662 IF-IC, IHC-P, W, IP H, M, R CD56 (NCAM) (123C3) Mouse mab 3576 IHC-P, W, F H Phospho-CREB (Ser133) (1B6) Mouse mab 9196 W H, M, R Phospho-CREB (Ser133) (87G3) Rabbit mab 9198 IF-IC, IF-F, IHC-P, W, F H, M, R Phospho-CREB (Ser133) Antibody 9191 IF-IC, IF-F, IHC-P, W, IP H, M, R CREB Antibody 9192 IF-IC, IHC-P, W, IP H, M, R, C CREB (48H2) Rabbit mab 9197 IF-IC, IF-F, IHC-P, W, IP, H, M, R, Mk CREB (86B10) Mouse mab 9104 IF-IC, IF-F, W, F H, M, R, Mk Phospho-Histone H3 (Ser10) (6G3) Mouse mab 9706 IF-IC, IF-F, W, IP, F H, M, R, Dr Phospho-Histone H3 (Ser10) Antibody 9701 IF-IC, IF-F, IHC-P, W, IP, F H, M, R, Mk, Dr Phospho-Histone H3 (Ser10) Antibody (Alexa Fluor 488 Conjugate) 9708 IF-IC, F H, M Phospho-Histone H3 (Ser10) Antibody (Alexa Fluor 647 Conjugate) 9716 F H, M Phospho-Histone H3 (Ser10) Antibody (Fluorescein Conjugate) 9704 IF-IC H, M, R, Dr Phospho-Histone H3 (Thr3) Antibody 9714 IHC-P, W H, M, R Histone H3 (3H1) Rabbit mab 9717 W H, M, Hm, Mk Histone H3 Antibody 9715 IF-IC, IHC-P, W H, M, R, Mk, (Dr) Phospho-HSP27 (Ser15) Antibody 2404 W H, Mk Phospho-HSP27 (Ser78) Antibody 2405 IHC-P, W H, Mk Phospho-HSP27 (Ser82) Antibody 2401 IF-IC, IHC-P, W H, Mk HSP27 (G31) Mouse mab 2402 IF-IC, IHC-P, W H, Mk c-jun (L70B11) Mouse mab 2315 IF-F, W, F H, M, R, Mk Phospho-p38 MAPK (Thr180/Tyr182) (3D7) Rabbit mab 9215 IF-IC, W, IP, F H, M, R, Mk Phospho-p38 MAPK (Thr180/Tyr182)(12F8) Rabbit mab (IHC Preferred) 4631 IF-IC, IHC-P, W, IP H, M, R, Mk Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab 9216 W, IP, F H, M, R Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab (Alexa Fluor 647 Conjugate) 4552 F H, M Phospho-p38 MAPK (Thr180/Tyr182) (28B10) Mouse mab (Alexa Fluor 488 Conjugate) 4551 F H, M Phospho-p38 MAP Kinase (Thr180/Tyr182) Antibody 9211 IF-IC, W, IP, F H, M, R p38 MAP Kinase (5F11) Mouse mab 9217 W, IP, F H, M, R p38 MAPK (5F11) Mouse mab (Alexa Fluor 647 Conjugate) 4555 F H p38 MAPK (5F11) Mouse mab (Alexa Fluor 488 Conjugate) 4556 F H p38 MAP Kinase (L53F8) Mouse mab 9228 W, IP, F H, M, R, Mk p38 MAP Kinase Antibody 9212 IHC-P, W, F H, M, R p38 MAP Kinase α Antibody 9218 IHC-P, W, IP H, M R, Mk p38 MAP Kinase δ Antibody 9214 W, IP H, M, R, Mk Phospho-p44/42 MAPK (Thr202/Tyr204) (20G11) Rabbit mab (IHC Preferred) 4376 IF-IC, IHC-P, W H, M, R Phospho-p44/42 MAPK (Thr202/Tyr204) (197G2) Rabbit mab 4377 IF-IC, W, F H, M, R Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab 9106 IF-IC, W, IP, F H, M, R, Hm, Z Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab (Alexa Fluor 488 Conjugate) 4374 F H, M Phospho-p44/42 MAPK (Thr202/Tyr204) (E10) Mouse mab (Alexa Fluor 647 Conjugate) 4375 F H, M Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody 9101 IF-IC, W, IP, F H, M, R, C, Hm, Z p44/42 MAP Kinase (137F5) Rabbit mab 4695 IHC-P, W, IP, F H, M, R, Mk p44/42 MAP Kinase (L34F12) Mouse mab 4696 IHC-P, W, IP, F H, M, R, Mk p44/42 MAP Kinase Antibody 9102 IHC-P, W, F H, M, R, Hm, Z Phospho-PTEN (Ser380) Antibody 9551 IF-IC, IF-F, IHC-P, W, IP H, M, R Phospho-PTEN (Ser380/Thr382/383) Antibody 9554 IF-IC, W, IP H, M, R PTEN (138G6) Rabbit mab 9559 IHC-P, W, F H, M, R, Mk PTEN (26H9) Mouse mab 9556 W, IP H, M, R, Hm, Mk PTEN Antibody 9552 W, IP H, M, R, Hm, Mk Phospho-SAPK/JNK (Thr183/Tyr185) Antibody 9251 IF-IC, IHC-P, W, IP, F H, M, R, Mk, (X) Phospho-SAPK/JNK(T183/Y185)(98F2) Rabbit mab 4671 W H, M, R Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mab 9255 W, IP, F H, M, R, (Dr) Phospho-SAPK/JNK (Thr183/Tyr185) (G9) Mouse mab (Alexa Fluor 647 Conjugate) 9257 F H, M SAPK/JNK (56G8) Rabbit mab 9258 W, IP H, M, R, Mk SAPK/JNK Antibody 9252 IF-IC, W, F H, M, R, Z XIAP Antibody 2042 IF-IC, W, IP, F H, M, R 18 Applications Reactivity IF-IC Immunofluorescence (Immunocytochemistry) IF-F Immunofluorescence (Frozen) IF-P Immunofluorescence (Paraffin) IHC-P Immunohistochemistry (Paraffin) W Western IP Immunoprecipitation F Flow cytometry E ELISA D DELFIA H Human M Mouse R Rat Hm Hamster Mk Monkey C Chicken X Xenopus All All species expected ( ) 100% sequence homology

19 Neuro_Broch all pgs 11/3/05 10:17 AM Page 19 Neuronal Injury and Response: Ischemia Signaling Ischemia (Stroke) Stress/Cell Death Survival/Regeneration Stem Cell Proliferation Sham 30 minutes Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mab (Alexa Fluor 488 Conjugate) #4584 Phospho-S6 Ribosomal Protein (Ser235/236) (2F9) Rabbit mab (Alexa Fluor 488 Conjugate) #4584 (green) and Phospho-Tyrosine Mouse mab (P-Tyr-100) (Alexa Fluor 647 Conjugate) #9415 (blue pseudocolor) staining rat striatum. Ischemia followed by 30 minutes or 4 hours reperfusion induces S6 and Tyrosine phosphorylation in Musashi-positive cells (red) in the subventricular zone. 4 hours Ischemic rat brain sections kindly provided by Dr Bingren Hu, University of Miami School of Medicine. 19

20 Neuro_Broch all pgs 11/3/05 10:17 AM Page 20 Histone H3 Notch Visit our website for frequent new product additions. Caspase-3 SAPK/JNK PRSRT STD U.S. Postage PAID??? c-jun MAP2 Akt APP/β-Amyloid CREB α-synuclein GSK-3β TrkA Opioid Receptor Presenilin PTEN 3 Trask Lane Danvers, MA USA p44/42 MAPK 20 Orders (toll free) CELL (2355) orders@cellsignal.com Fax ordering Technical support (toll-free) TECH (8324) support@cellsignal.com MAPK Synapsin Dab1 Cleaved Caspase-3