Anti p ripk1

Heart tissues and cells were homogenized and sonicated in a RIPA lysis buffer (Beyotime, Shanghai, China), including a protease inhibitor cocktail. Target proteins were separated by SDS/PAGE gels, before being transferred onto 0.22-μm PVDF membranes. After blocking with 5% bovine serum albumin (BSA), PVDF membranes were incubated with primary antibodies, including anti-Vinculin (Abcam, #ab219649), anti-FUNDC1(Abcam, #ab224722), anti-AIM2 (Abcam, #ab204995), anti-ZBP1 (Abcam, #ab290736), anti-Pyrin (Abcam, #ab195975), anti-Caspase1 (Abcam, #ab207802), anti-Caspase3 (Abcam, #ab32351), anti-Caspase8 (Proteintech, 13423-1-AP), anti-GSDMD (Abcam, #ab209845), anti-MLKL (Abcam, #ab184718), anti-p-MLKL (Abcam, #ab196436) anti-RIPK1 (Cell Signaling Technology, #73271), anti-p-RIPK1 (Cell Signaling Technology, #44590), anti-RIPK3 (Cell Signaling Technology, #10188), anti-p-RIPK3 (Cell Signaling Technology, #93654), anti-p-TUFM antibodies (Abcam, #ab173300) and anti-Flag antibodies (Cell Signaling Technology, #14793) overnight at 4 °C. Secondary antibodies were employed for membrane incubation. Films were scanned and detected with a Bio-Rad calibrated densitometer.