Western Blot Protein Analysis Protocol

The extraction of protein was performed as described^{76 (link)}. Proteins were separated by 12% Bis-Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA) by 350 mA for 90 min, and blocked for 1 hour at room temperature. Primary antibodies used to probe blots were mouse anti-TICAM-1 (sc-514384, mAb, Santa, USA), mouse anti-FLIPS/L (sc-5276, mAb, Santa, USA), rabbit anti-cleaved caspase-8 (9496, mAb, CST, USA), rabbit anti-total caspase-8 (ab108333, mAb, Abcam, USA), rabbit anti-RIPK1/RIP1 (NBP1-77077, polyclonal Ab, Novus, USA), rabbit anti-RIP3 (ab152130, polyclonal Ab, Abcam, USA), rabbit anti-MLKL (ab184718, mAb, Abcam, USA), rabbit anti-p-MLKL (phospho S358) (ab187091, mAb, Abcam, USA), rabbit anti-TOP1 (20705-1-AP, polyclonal Ab, Proteintech, USA), rabbit anti-BET (13232, mAb, CST, USA), rabbit anti-CDK9 (2316, mAb, CST, USA) and rabbit anti-GAPDH (MB001, mAb, Bioworld, USA), which were incubated overnight at 4 °C. Subsequently, HRP-conjugated secondary antibodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 hour. Blots were then visualized by a chemiluminescent imaging system (Tanon, Shanghai, China).