Enhanced chemiluminescence luminescence reagent

Protein extracts were separated on 8–15% polyacrylamide gels and then transferred onto polyvinylidene difluoride (PVDF) membranes (MilliporeSigma, Burlington, MA, USA). After blocking nonspecific binding with 5% nonfat dry milk (Sangon Biotech Shanghai Co., Ltd.) membranes were probed with primary antibodies overnight at 4°C and incubated with ppropriate HRP-labeled secondary antibodies at room temperature for 1 h. Membranes were then visualized with an enhanced chemiluminescence luminescence reagent (Meilunbio). The intensities of the bands were determined by ImageJ Launcher broken symmetry software program (NIH, Bethesda, MD, USA). Following antibodies were used: rabbit anti-mouse cleaved caspase-8 (#8592; Cell Signaling Technology), rabbit anti caspase-8 (AC056; Beyotime), Phospho-RSK2 (Ser227) Rabbit mAb (#3556S; Cell Signaling Technology), PDK1 (D37A7) Rabbit mAb (#5662S; Cell Signaling Technology), Caspase-3 (D3R6Y) Rabbit mAb (#14220S; Cell Signaling Technology), Caspase-1 (D7F10) Rabbit mAb (#3866S; Cell Signaling Technology), Anti-MLKL (phospho S345) (ab196436; Abcam), Anti-MLKL (phospho S358) (ab187091; Abcam), Anti-pro Caspase-1 + p10 + p12 (ab179515; Abcam), Anti-GSDMD antibody (ab209845; Abcam), Anti-GSDMD antibody (ab225867; Abcam), Anti-cleaved N-terminal GSDMD (ab215203; Abcam), anti-beta-actin (bs-0061 R; Bioss).

Cells were lysed in RIPA Buffer (Beyotime) containing a protease and phosphatase inhibitor mini tablet (Thermo Fisher Scientific, Waltham, MA, USA). After normalization of protein concentrations with a BCA assay kit (Beyotime), samples were separated by SDS PAGE and transferred onto polyvinylidene fluoride membranes (Millipore, Saint Louis, Missouri, USA). Membranes were blocked in 5% nonfat milk in TBST and probed with primary antibodies against p-P65 (Abcam, Cambridge, MA, USA), P65 (ImmunoWay, Plano, TX, USA), IKBα (Santa Cruz Biotechnology, Dallas, TX, USA), IKKβ (Proteintech, Rosemont, IL, USA), p-IKKβ (Cell Signaling Technology, Danvers, MA, USA), KEAP1 (Proteintech), FLAG (ImmunoWay), EGFP (Bioss, Beijing, China), HA (Proteintech), α-tubulin (Proteintech), β-ACTIN (Bioss), and GAPDH (Bioss). Primary antibodies were incubated overnight at 4°C, washed, and incubated for 1 h with HRP-coupled secondary antibodies at room temperature. The blots were then visualized with an enhanced chemiluminescence luminescence reagent (Meilunbio, Dalian, China). The amount of correlated proteins was analyzed by Image J software program.