Ripk1

RIPK3 (1:2000 cat# NBP1-77299, Novus Biologicals, Centennial, CO, USA), internal control GAPDH (1:100,000, Cat# MAB374, Sigma-Aldrich, St Louis, MO, USA), β-ACTIN (1:2000, Cat# 4967, Cell Signaling Technology, Inc., Danvers, MA, USA), RIPK1(1:1000, Cat# MAB3585, R&D systems, Inc., Minneapolis, MN, USA), HIF1α (1:1000, Cat# 3716, Cell Signaling Technology, Inc., Danvers, MA, USA), cleaved CASPASE3 (1:5000, Cat# 9664, Cell Signaling Technology, Inc., Danvers, MA, USA), MLKL (1:2000, Cat# MABC604, Millipore Corp, Burlington, MA, USA), CASPASE1 (1:100, Cat# sc-56036, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and CASPASE8 (1:100, Cat# sc-81656, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) primary antibodies were used for the study. Horseradish peroxidase (HRP) conjugated goat anti-rabbit secondary antibody (Cat# 656120, Invitrogen, Thermo Fisher Scientific, Hanover Park, IL, USA) was used for cleaved CASPASE3 (1:100,000), RIPK3, HIF1α, CASPASE8, CASPASE1 (1:20,000), goat anti-rat secondary antibody (Cat# 629320, Invitrogen, Thermo Fisher Scientific, IL, USA) was used for MLKL (1:20000) and goat anti-mouse secondary antibody (Cat# G21040, Invitrogen, Thermo Fisher Scientific, IL, USA) was used for RIPK1 (1:20,000) and GAPDH (1:100,000).

Tumor tissue or cells were homogenized and lysed in RIPA buffer supplemented with a protease inhibitor cocktail and Phenylmethylsulfonyl Fluoride (Sigma). Protein samples were separated by SDS-PAGE and transferred onto PVDF membranes (Bio-Rad). Monoclonal/polyclonal primary antibodies and appropriate HRP-conjugated secondary antibodies were employed for Western blotting. Protein visualization was achieved using ECL-Plus. For immunoprecipitation (IP), cells were lysed, and protein concentrations were quantified. Primary antibodies were added and incubated overnight at 4 °C with rotation. Protein G beads were added and incubated for 3 h at 4 °C with rotation. The resin was centrifuged, washed three times with lysis buffer, and analyzed by immunoblotting.
The following antibodies were used: TRIM28 (Cat: PA5-27648, Invitrogen), RIPK1 (Cat: PA5-20811, Invitrogen), Phospho-IκBα (Cat: #2859, Cell Signaling Technology), IκBα (Cat: #4814, Cell Signaling Technology), Phospho-IKKα/β (Cat: #2697, Cell Signaling Technology), IKKα (Cat: #2682, Cell Signaling Technology), IKKβ (Cat: #8943, Cell Signaling Technology), anti-Flag (Cat: #14,793, Cell Signaling Technology), anti-Ubiquitin (Cat: #3936, Cell Signaling Technology), anti-K48-linkage Specific Polyubiquitin (Cat: #8081, Cell Signaling Technology), and anti-K63-linkage Specific Polyubiquitin (Cat: #5621, Cell Signaling Technology).