Nonfat dry milk

Thirty milligrams of the ceca was cut into pieces and lysed in 1 ml of RIPA buffer containing protease inhibitors and phosphatase inhibitors (Beyotime Biotechnology). Samples were homogenized on ice, centrifuged for supernatant at 12,000 rpm for 30 min at 4°C, and heated to 100°C for 5 min. Protein extracts resuspended in sample loading buffer were separated by electrophoresis through 12–15% polyacrylamide gels. Following electrophoretic transfer of proteins onto polyvinylidene difluoride (PVDF) membranes (Millipore), non-specific binding was blocked by incubation with 5% non-fat dry milk (Sangon Biotech Shanghai Co., Ltd.), and then membranes were incubated with primary antibodies anti-phospho-ERK1/2 and anti-phospho-JNK1/2 (1:1,000 dilution, Cell Signaling Technology); anti-NLRP3, anti-NLRC4, anti-Caspase-1, anti-Caspase-11, anti-GSDMD, and anti-histone H3 (1:1,000 dilution, abcam); anti-GAPDH (1:1,000 dilution, Boster); and anti-Tubulin (1:1,000, Beyotime) overnight at 4°C. Membranes were then washed and incubated with the horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG (1:3,000 dilution, Beyotime) for 1 h at room temperature. Proteins were visualized using enhanced chemiluminescence (ECL) reagent (Meilunbio). The grayscale values of the bands were determined by ImageJ launcher broken symmetry software program (National Institutes of Health, Bethesda, MD, USA).