Journal: Cell Death & Disease
Article Title: Redundant and receptor-specific activities of TRADD, RIPK1 and FADD in death receptor signaling
Figure Lengend Snippet: Relevance of TRADD, RIPK1, and FADD for caspase activation and cell death induction by TNF and TRAIL. a Western blot evaluation of TRADD, RIPK1, and FADD expression of HeLa-RIPK3 con , HeLa-RIPK3-TRADD KO , HeLa-RIPK3-RIPK1 KO , and HeLa-RIPK3-FADD KO cells. fl full-length. b The various HeLa-RIPK3 variants were stimulated in technical triplicates as indicated with TNF (100 ng/ml), TRAIL (100 ng/ml), CHX (2.5 µg/ml), ZVAD (Z, 20 µM), and nec1 (N, 90 µM). The next day, cellular viability was evaluated by crystal violet staining. A representative panel of experiments is shown. For statistical analysis of independent experiments please see Tables 1 and 2 and Supplementary Tables I – III . c Western blot analysis of phosphorylated RIPK1 in HeLa-RIPK3 and HeLa-RIPK3-FADD KO cells treated for 2, 4, or 8 h with 100 ng/ml of TNF or TRAIL. Where indicated cells were challenged in the presence of CHX (2.5 µg/ml) and ZVAD (20 µM). d TNFR1- and TRAIL death receptor-associated signaling complexes were immunoprecipitated from the various HeLa-RIPK3 variants with a TNFR1-specific Fc fusion protein of TNF or Fc-TRAIL and protein G beads. IPs were analyzed by western blotting for the presence of the indicated proteins. For western blot analysis of lysates see Supplementary Data (Fig. S5A). e HeLa-RIPK3 variants were stimulated overnight in the presence of 2.5 µg/ml CHX with 1, 10, or 100 ng/ml of TNF or TRAIL. Total cell lysates were analyzed by western blot. f HeLa-RIPK3 con and HeLa-RIPK3-TRADD KO cells were challenged in technical triplicates with increasing concentrations of TNF or TRAIL in the presence of the indicated mixtures of CHX (C, 2.5 µg/ml), nec1 (N, 90 µM, apoptotic conditions), and ZVAD (Z, 20 µM, necroptotic conditions). Cellular viability was determined the next day by crystal violet staining
Article Snippet: Following primary antibodies have been used: anti-caspase-8 (Santa Cruz, E-20, sc-6133), anti-caspase-8 (Enzo, 5F7), anti-caspase-3 (Cell Signaling, 8G10), anti-caspase-9 (Cell Signaling, # 9502), anti-PARP (BD Biosciences), anti-CYLD (Cell Signaling, D1A10), anti-DR4/TRAILR1 (Cell Signaling, D9S1R), anti-DR5/TRAILR2 (Cell Signaling, D4E9), anti-phospho-RIPK1 (Ser166) (Cell signaling, D1L3S), which is specific for serine 166 phosphorylated necroptosis-competent RIPK1, anti-RIPK1 (Cell Signaling, D94C12), anti-RIPK1 (BD Biosciences, #610459), anti-tubulin (ThermoFisher Scientific (DM1A), anti-TNFR1 (Cell Signaling, C25C1), anti-TRADD (Cell Signaling, 7G8), anti-A20 (Cell Signaling, D13H3), anti-IKKß (Cell Signaling, D30C6), anti-FADD (Cell Signaling, #2782), anti-Sharpin (Abcam ab125188), anti-TRAF2 (Santa Cruz, C-20, sc-876), anti-FLIP (Biomol, NF6, AG-20B-0056), anti-phospho-IκBα (Ser32) (Cell Signaling, 14D4), which recognizes serine 32 phosphorylated IκBα prone for ubiquitination and proteasomal degradation, and anti-IκBα (Cell Signaling, L35A5).
Techniques: Activation Assay, Western Blot, Expressing, Staining, Immunoprecipitation