Complete mini protease inhibitor cocktail and phosphate inhibitors

The Western blotting analysis was conducted as previously described [27 ]. Briefly, cultured cells, tumors and normal mucosa from the buccal mucosa and tongue were collected from mice, then the protein lysates were generated using M-PER or RIPA reagent (Pierce, Rockford, IL) containing a complete mini protease inhibitor cocktail and phosphate inhibitors (Roche, Branchburg, NJ). After denaturation the total protein was separated using 10% SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore). The blots were then blocked with 5% non-fat dry milk at room temperature for 1 h, and incubated overnight with the corresponding primary antibodies at dilutions recommended by the suppliers at 4 °C, finally by incubation with horseradish peroxidase-conjugated secondary antibody (Pierce, Rockford, IL). Next, the blots were detected using an enhanced chemiluminescence detection kit (West Pico, Thermo). GAPDH was detected on the same membrane and used as a loading control.

Western blot analysis was performed as previously reported.^[29] After each treatment, mouse tumors were lysed using radio immunoprecipitation assay (RIPA) reagent (Pierce, Rockford, IL, USA) containing a complete miniprotease inhibitor cocktail and phosphate inhibitors (Roche, Branchburg, NJ, USA).Proteins were separated using 10% sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore).Then, the protein was incubated with anti‐GSDME (Abcam, ab215191) and anti‐β‐actin antibody (Abcam, ab8226).