Anti rabbit and anti mouse antibodies

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).

Western blotting was performed as we described before^{13 (link)}. Briefly, cells were washed with PBS and lysed. The concentration of total proteins was determined using a Nanodrop (Thermo Fisher, USA). Proteins were separated by 12% Bis–Tris Plus gels (Genscript, China), transferred to PVDF membrane (Millipore, USA), and blocked for 1 h at room temperature and incubated with primary antibodies, anti-transferrin receptor (TfR) (1:1000; ab214039, Abcam, USA), anti-NCOA4 (1:1000; A5695, ABclonal, China), anti-ferritin (1:1000; ab65080, Abcam), anti-ferroportin (FPN) (1:1000; ab78066, Abcam), anti-LC3B (1:1000; 83506, CST, USA), anti-HIF-1α (1:1000; ab2185, Abcam), anti-CDK9 (1:1000;2316,CST), anti-BRD4 (1:1000; ab128874, Abcam), anti-p38 (1:1000; 8690, CST), anti-Phospho-p38 (1:1000; 4511, CST), anti-JNK (1:1000; 9252, CST), anti-Phospho-JNK (1:1000; 4668, CST), anti-ERK1/2 (1:1000; 4695, CST), anti-Phospho-ERK1/2 (1:1000; AF1015, Affinity, China), or GAPDH (1:1000; MB001, Bioworld, USA). Subsequently, HRP-conjugated secondary anti-bodies including anti-rabbit and anti-mouse antibodies (Fcmacs, China) were incubated for 1 h. Blots were then visualized by a chemiluminescent imaging system (Tanon, China). The optical density of each lane was read using ImageJ (Bethesda, USA).