Western Blotting Standardized Protocols

Western blotting was performed using the standard procedures as we previously reported^{58 (link),59 (link)}. Proteins were extracted from cells or tissues using RIPA lysis buffer (Solarbio). Equal volumes of lysates were loaded and separated on 10% SDS-PAGE gels and blotted on polyvinylidene difluoride membrane. After being blocked with 5% skim milk for 2 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 ˚C. The primary antibody used in western blot analysis included Anti-ASC (1:1000; ab155970; Abcam); anti-caspase-1 (1:1000; ab207802; Abcam); anti-caspase-4 (1:1000; ab238124; Abcam); anti-GSDMD (1:1000; 66387-1-Ig; Proteintech); anti-PD1 (1:1000; ab52587; Abcam) and anti-GAPDH (1:500; ab8245; Abcam;). Anti-rabbit-HRP (1:5000; #7074, Cell Signaling Technology) and anti-mouse-HRP (1:5000; #7076, Cell Signaling Technology) were used as secondary antibodies and incubated at room temperature for 1 h. The blots were detected using a chemiluminescence kit (cat. no. 34577; Thermo Fisher Scientific, Inc.) and imaged using MiniChemi 610 system (Sage Creation Science, Co., Ltd.).