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8 Figure S1: NIK-deficient cells resist to Tweak/TNFα-induced cell death (a) Two independent clones of NIK WT and NIK KO MEFs were treated for 24 hours with Tweak (200 ng/ml) and TNFα (200 U/ml) and cell survival was measured with Cell Titer-Glo kit. (b) aly/aly homozygote MEFs were treated and analyzed like in (a). Figure S2: NIK/RIP1-mediated caspase activation acts through the mitochondrial pathway (a) RIP1 +/+ and RIP1 -/- MEFs were treated for 24 hours with or without TNFα (200 U/ml) and increasing amount of the Smac mimetics CmpA (250 nm), and cell survival was measured with Cell Titer-Glo kit. (b) NIK +/+ and NIK -/- MEFs were treated as indicated and protein extracts were analyzed by Western-blot for phosphor- JNK expression. (c) Independent WT and KO MEFs for NIK were tested for their expression level of c-flip by Western-blot. (d) NIK KO MEFs reconstituted with empty vector (EV), NIK WT (WT) or NIK kinase-dead (KD) were analyzed for their c-flip content (e) DEVDase activity of Bax/Bak +/+ and Bax/Bak -/- MEFs treated for various time with two doses of Tweak and a fixed concentration of TNFα (200 U/ml). (f) Bax/Bak +/+ and Bax/Bak -/- MEFs were treated with Tweak (200 ng/ml) and TNFα (200 U/ml) for different time periods. Levels of cleaved caspase-8 and cleaved caspase-3 were assessed by Western-blot. Statistical analyses: *** P< Figure S3: RIP1 is a substrate of NIK but its interaction does not require the RHIM domain
9 (a) HEK293T cells were transfected with GFP-RIP3 and FL kinase dead RIP1-V5 or ΔRHIM RIP1-V5 expression vectors. RIP3-GFP complexes were immunoprecipitated using an anti-gfp antibody and RIP1 interactions were revealed by Western-blot with an anti-rip1 antibody. (b) Specificity of recombinant full-length RIP1was assessed by in vitro kinase assay using RIP1 kinase inhibitor Nec-1. (c) Specificity of recombinant NIK kinase domain was assessed by in vitro kinase assay using NIK kinase inhibitor and/or RIP1 kinase inhibitor Nec-1. (d) NIK +/+ and NIK -/- MEFs were left untreated or treated with a mix of TNFα and Smac mimetics (CmpA) and the phosphorylation status of RIP1 was analyzed by Western-blot with cell extracts treated or not with lambda phosphatase (λ phos.). (e) In vitro kinase assay with active full-length RIP1 in the presence of active recombinant NIK and the RIP1 kinase inhibitor Nec-1. (f) In vitro kinase assay with inactive full-length RIP1 in the presence of active recombinant NIK. Figure S4: Smac mimetics/tnfα-induced complex IIb assembly is defective in NIKdeficient MEFs (a) NIK +/+ and NIK -/- MEFs were treated with CmpA and/or TNFα for the indicated time periods prior to an immunoprecipitation with an anti-caspase-8 antibody and detection of both caspase-8 and RIP1 by Western-blot. (b) Expression profile of death domain-containing proteins in NIK +/+ and NIK -/- MEFs treated with Tweak/TNFα in the presence of z-vad-fmk (TTZ). (c) Level of c-iap1/2 depletion in NIK +/+ and NIK -/- MEFs treated with CmpA, ns, non-specific signal. (d) NIK +/+ MEFs were treated with Tweak/TNFα/Z-VAD for 60 min. prior to immunoprecipitation with irrelevant or anti-nik antibodies. The immunoprecipitates were subjected to in vitro caspase-8 activity measurement. P value was 0,02.
10 Figure S5: TNFR1/LTβR-induced thymus involution and cell death is blocked in NIK knock-out mice. (a) Images of thymi from postnatal (8 weeks) BM reconstitution experiments. Lethally irradiated (1100 rad) WT and NIK -/- recipient mice were reconstituted with BM from Tg Lck-LTαβ mice. (b) Quantitative analyses of relative thymus weight of reconstitution experiments achieved in (a). (c) Analysis of thymic architecture (H&E staining) of the indicated post-reconstitution (5-6 weeks) experiments and cleaved caspase-3 staining. Statistical analyses: *** P< Figure S6: p100-deficiency does not prevent Tweak/TNFα-induced apoptosis. MEFs deficient for p100 treated with Tweak and TNFα displays the apoptotic cleavage of caspase-8 and caspase-3. Figure S7: RIP1 and NIK mediate Adenoviral/TNFα-induced hepatocyte caspase-3 cleavage. (a, b) Dosage of serum ALT and serum AST in aly/wt (n=3) and aly / aly (n=3) mice treated for 4 hours with D-Gal-N and TNFα. (c, d) Dosage of serum ALT and serum AST in WT mice treated for 4 hours with D-Gal-N and TNFα in the presence or absence of Nec-1 (n=3 for each treatment).
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doi:10.1038/nature09732 Supplementary Figure 1: Depletion of Fbw7 results in elevated Mcl-1 abundance. a, Total thymocytes from 8-wk-old Lck-Cre/Fbw7 +/fl (Control) or Lck-Cre/Fbw7 fl/fl (Fbw7 KO) mice
Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the
Supplementary Fig. 1. Schematic structure of TRAIP and RAP80. The prey line below TRAIP indicates bait and the two lines above RAP80 highlight the prey clones identified in the yeast two hybrid screen.
SUPPLEMENTARY INFORMATION FIGURE LEGENDS
SUPPLEMENTARY INFORMATION FIGURE LEGENDS Fig. S1. Radiation-induced phosphorylation of Rad50 at a specific site. A. Rad50 is an in vitro substrate for ATM. A series of Rad50-GSTs covering the entire molecule
Supplementary Figure 1 (related to Figure 1) a. SVEC cells stably expressing egfp tbid 2A BCL xl were analysed by Western blot for expression of egfp
Supplementary Figure 1 (related to Figure 1) a. SVEC cells stably expressing egfp tbid 2A BCL xl were analysed by Western blot for expression of egfp tbid and BCL xl. TOM20 was used as a loading control.
Supplemental Information. The TRAIL-Induced Cancer Secretome. Promotes a Tumor-Supportive Immune. Microenvironment via CCR2
Molecular Cell, Volume 65 Supplemental Information The TRAIL-Induced Cancer Secretome Promotes a Tumor-Supportive Immune Microenvironment via CCR2 Torsten Hartwig, Antonella Montinaro, Silvia von Karstedt,
Cell death analysis using the high content bioimager BD PathwayTM 855 instrument (BD
Supplemental information Materials and Methods: Cell lines, reagents and antibodies: Wild type (A3) and caspase-8 -/- (I9.2) Jurkat cells were cultured in RPMI 164 medium (Life Technologies) supplemented
Supplementary Figure 1. α-synuclein is truncated in PD and LBD brains. Nature Structural & Molecular Biology: doi: /nsmb.
Supplementary Figure 1 α-synuclein is truncated in PD and LBD brains. (a) Specificity of anti-n103 antibody. Anti-N103 antibody was coated on an ELISA plate and different concentrations of full-length
Supplementary Information
Supplementary Information Supplementary Figures Supplementary Figure 1. MLK1-4 phosphorylate MEK in the presence of RAF inhibitors. (a) H157 cells were transiently transfected with Flag- or HA-tagged MLK1-4
Supplementary Materials
Supplementary Materials Supplementary Figure 1. PKM2 interacts with MLC2 in cytokinesis. a, U87, U87/EGFRvIII, and HeLa cells in cytokinesis were immunostained with DAPI and an anti-pkm2 antibody. Thirty
SUPPLEMENTARY INFORMATION. Small molecule activation of the TRAIL receptor DR5 in human cancer cells
SUPPLEMENTARY INFORMATION Small molecule activation of the TRAIL receptor DR5 in human cancer cells Gelin Wang 1*, Xiaoming Wang 2, Hong Yu 1, Shuguang Wei 1, Noelle Williams 1, Daniel L. Holmes 1, Randal
Supplementary Figure 1: Overexpression of EBV-encoded proteins Western blot analysis of the expression levels of EBV-encoded latency III proteins in
Supplementary Figure 1: Overexpression of EBV-encoded proteins Western blot analysis of the expression levels of EBV-encoded latency III proteins in BL2 cells. The Ponceau S staining of the membranes or
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Dynamic Phosphorylation of HP1 Regulates Mitotic Progression in Human Cells Supplementary Figures Supplementary Figure 1. NDR1 interacts with HP1. (a) Immunoprecipitation using
GFP CCD2 GFP IP:GFP
D1 D2 1 75 95 148 178 492 GFP CCD1 CCD2 CCD2 GFP D1 D2 GFP D1 D2 Beclin 1 IB:GFP IP:GFP Supplementary Figure 1: Mapping domains required for binding to HEK293T cells are transfected with EGFP-tagged mutant
Li et al., Supplemental Figures
Li et al., Supplemental Figures Fig. S1. Suppressing TGM2 expression with TGM2 sirnas inhibits migration and invasion in A549-TR cells. A, A549-TR cells transfected with negative control sirna (NC sirna)
Reviewers' Comments: Reviewer #2 (Remarks to the Author)
Reviewers' Comments: Reviewer #2 (Remarks to the Author) The manuscript of Zhang et al. describes the role of RIP1 phosphorylation in the necrosome assemblage. Taking into account that the role of RIP1
T H E J O U R N A L O F C E L L B I O L O G Y
T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Nakajima and Tanoue, http://www.jcb.org/cgi/content/full/jcb.201104118/dc1 Figure S1. DLD-1 cells exhibit the characteristic morphology
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/5/233/ra50/dc1 Supplementary Materials for Epidermal Growth Factor Receptor Is Essential for Toll-Like Receptor 3 Signaling Michifumi Yamashita, Saurabh Chattopadhyay,
At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in
Supplementary Materials and Methods Barrier function assays At E17.5, the embryos were rinsed in phosphate-buffered saline (PBS) and immersed in acidic X-gal mix (100 mm phosphate buffer at ph4.3, 3 mm
Supplementary Materials for
www.sciencesignaling.org/cgi/content/full/2/85/ra48/dc1 Supplementary Materials for The VDAC2-BAK Rheostat Controls Thymocyte Survival Decheng Ren, Hyungjin Kim, Ho-Chou Tu, Todd D. Westergard, Jill K.
Supplementary Fig. 1 Kinetics of appearence of the faster migrating form of Bcl-10.
α-cd3 + α-cd28: Time (min): + + + + + + + + + 0 5 15 30 60 120 180 240 300 360 360 n.s. Supplementary Fig. 1 Kinetics of appearence of the faster migrating form of. Immunoblot of lysates from Jurkat cells
Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in
Supplementary information Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer s disease Zhentao Zhang, Mingke Song, Xia Liu, Seong Su Kang, Il-Sun Kwon, Duc
3 P p25. p43 p41 28 FADD. cflips. PE-Cy5 [Fluorescence intensity]
![3 P p25. p43 p41 28 FADD. cflips. PE-Cy5 [Fluorescence intensity]](/thumbs/87/97394430.jpg "3 P p25. p43 p41 28 FADD. cflips. PE-Cy5 [Fluorescence intensity]")
L S p4 3 D3 76 N S L Ve ct or p4 3 D3 76 N S L Ve ct or A aspase 8 FADD TRAF2 D95-R - + Vector D95L TL I S L D376N T RAIL-R1 T RAIL-R2 D95-R E-y5 [Fluorescence intensity] Supplemental Fig. 1 Different
Supplemental Materials and Methods
Supplemental Materials and Methods Co-immunoprecipitation (Co-IP) assay Cells were lysed with NETN buffer (20 mm Tris-HCl, ph 8.0, 0 mm NaCl, 1 mm EDT, 0.5% Nonidet P-40) containing 50 mm β-glycerophosphate,
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Figure S1: Activation of the ATM pathway by I-PpoI. A. HEK293T cells were either untransfected, vector transfected, transfected with an I-PpoI expression vector, or subjected to 2Gy γ-irradiation. 24 hrs
Figure 1: Caspase activation during LPS-induced macrophage apoptosis. J774A.1
Supplementary material Figure 1: Caspase activation during LPSinduced macrophage apoptosis. J774A.1 macrophagelike cells were incubated with LPS plus SB202190 (10 µm), whereas NIH3T3 fibroblasts were incubated
Fig. S1 TGF RI inhibitor SB effectively blocks phosphorylation of Smad2 induced by TGF. FET cells were treated with TGF in the presence of
Fig. S1 TGF RI inhibitor SB525334 effectively blocks phosphorylation of Smad2 induced by TGF. FET cells were treated with TGF in the presence of different concentrations of SB525334. Cells were lysed and
Supplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. CD11b expression on different B cell subsets in anti-snrnp Ig Tg mice. (a) Highly purified follicular (FO) and marginal zone (MZ) B cells were sorted from
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3240 Supplementary Figure 1 GBM cell lines display similar levels of p100 to p52 processing but respond differentially to TWEAK-induced TERT expression according to TERT promoter mutation
* ** ** * IB: p-p90rsk. p90rsk (Ser380) (arbitrary units) (Ser380) p90rsk. IB: p90rsk. Tubulin. IB: Tubulin. Ang II (200 nm) Ang II (200 nm)
I: p-p9rsk I: p9rsk I: C I: p-p9rsk I: p9rsk 5 (ka) 5 5 (min) Ang II ( nm) p-p9rsk (Ser8) p9rsk p-p9rsk (Ser8) p9rsk (h) Mannitol 5 mm -Glucose 5 mm p9rsk (Ser8) (arbitrary units) p-p9rsk (Ser8) (arbitrary
SUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3209 Supplementary Figure 1 IR induces the association of FH with chromatin. a, U2OS cells synchronized by thymidine double block (2 mm) underwent no release (G1 phase) or release for 2
Supplementary Figure 1. The Hsp70 acetylation level is related to the co-chaperone binding of Hsp70 under various stress conditions.
Supplementary Figure 1. The Hsp70 acetylation level is related to the co-chaperone binding of Hsp70 under various stress conditions. 1 (a) Etoposide treatment gradually changes acetylation level and co-chaperone
(A-B) P2ry14 expression was assessed by (A) genotyping (upper arrow: WT; lower
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Figure S1 (related to Fig. 1): The prototypical mitochondrial pathway of apoptosis is involved in cell-death of v-src-transformed cells.
Figure S1 (related to Fig. 1): The prototypical mitochondrial pathway of apoptosis is involved in cell-death of v-src-transformed cells. (A) Non-transformed (Control cells) and v-srctransformed 3T3 cells
Reagents and cell culture Mcl-1 gene expression: real-time quantitative RT-PCR In vitro PP2A phosphatase assay Detection of Mcl-1 in vivo
Reagents and cell culture Antibodies specific for caspase 3, PARP and GAPDH were purchased from Cell Signaling Technology Inc. (Beverly, MA). Caspase inhibitor z-vad-fmk and ROS scavenger N-acetyl-Lcysteine
SUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Supplementary figures Supplementary Figure 1: Suv39h1, but not Suv39h2, promotes HP1α sumoylation in vivo. In vivo HP1α sumoylation assay. Top: experimental scheme. Middle: we
Supplementary Figure 1. IFN-γ induces TRC dormancy. a, IFN-γ induced dormancy
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Figure S1 The effect of T198A mutation on p27 stability. a, Hoechst 33342 staining for nuclei (see Fig 1d). Scale bar, 100 μm. b, Densitometric analysis of wild type and mutant p27 protein levels represented
Supplementary Table 1. The Q-PCR primer sequence is summarized in the following table.
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Supplementary Figure 1. Bone density was decreased in osteoclast-lineage cell specific Gna13 deficient mice. (a-c) PCR genotyping of mice by mouse
Supplementary Figure 1. Bone density was decreased in osteoclast-lineage cell specific Gna13 deficient mice. (a-c) PCR genotyping of mice by mouse tail DNA. Primers were designed to detect Gna13-WT/f (~400bp/470bp)
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Supplemental Materials and Methods Short hairpin RNA (shrna) against MMP14. Lentiviral plasmids containing shrna (Mission shrna, Sigma) against mouse MMP14 were transfected into HEK293 cells using FuGene6
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(Supplementary Methods and Materials) GST pull-down assay GST-fusion proteins Fe65 365-533, and Fe65 538-700 were expressed in BL21 bacterial cells and purified with glutathione-agarose beads (Sigma).
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Supplementary Figure 1. APP cleavage assay. HEK293 cells were transfected with various
Supplementary Figure 1. APP cleavage assay. HEK293 cells were transfected with various GST-tagged N-terminal truncated APP fragments including GST-APP full-length (FL), APP (123-695), APP (189-695), or
FSC-H FSC-H FSC-H
Supplement Figure A 4 h control + h B 4 h control + h Supplement Figure A-H BV6/TNF-α + ctrl A B 9 h C D h E F 8 h G H Supplement Figure I-P + ctrl I J 8 h K L 4 h M N h O P Supplement Figure 3 A Survival
Figure 1: TDP-43 is subject to lysine acetylation within the RNA-binding domain a) QBI-293 cells were transfected with TDP-43 in the presence or
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DOI: 10.1038/ncb2743 Figure S1 stabilizes cellular protein level, post-transcriptionally. (a, b) and DDR1 were RNAi-depleted from HEK.293.-CBG cells. Western blots with indicated antibodies (a). RT-PCRs
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DOI: 1.138/ncb3342 EV O/E 13 4 B Relative expression 1..8.6.4.2 shctl Pcdh2_1 C Pcdh2_2 Number of shrns 12 8 4 293T mterc +/+ G3 mterc -/- D % of GFP + cells 1 8 6 4 2 Per2 p=.2 p>
SureSilencing sirna Array Technology Overview
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ASPP1 Fw GGTTGGGAATCCACGTGTTG ASPP1 Rv GCCATATCTTGGAGCTCTGAGAG
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Supplementary Figure 1
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Supplementary Fig. 1 Proteomic analysis of ATR-interacting proteins. ATR, ARID1A and
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Supplementary Figure 1 a
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Product Line Overview
Product Line Overview Your Toolbox for Apoptosis and Necrosis Measurement Product Line Products for accurate quantification of Apoptosis and Necrosis The PEVIVA Product Line is manufactured by VLVbio,
Supplementary information
Supplementary information The E3 ligase RNF8 regulates KU80 removal and NHEJ repair Lin Feng 1, Junjie Chen 1 1 Department of Experimental Radiation Oncology, The University of Texas M. D. Anderson Cancer
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Supplementary Figure 1
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Supporting Information He et al. 10.1073/pnas.1116302108 SI Methods Cell Culture. Mouse J774A.1 and RAW 264.7 macrophages were obtained from ATCC and were cultured in MEM supplemented with 10% FS (Sigma)
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b alternative classical none
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Supporting Information
Supporting Information Üstün and Börnke, 2015 Figure S1: XopJ does not display acetyltransferase activity. Autoacetylation activity in vitro. Acetylation reactions using MBP, MBP-XopJ, GST and GST-HopZ1a
Nature Medicine doi: /nm.3554
SUPPLEMENTARY FIGURES LEGENDS Supplementary Figure 1: Generation, purification and characterization of recombinant mouse IL-35 (ril-35). High-Five insect cells expressing high levels of the bicistronic
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