The transfected cells were collected after 48 h, and RIPA cell lysate was added. Total cell proteins were extracted on ice, and then the protein concentration was detected by a BCA protein detection kit. The protein sample was heated and denaturized in a boiling water bath. The denaturized protein sample of the equal amount was added to the loading hole for SDS-PAGE gel electrophoresis. After the protein was separated, it was transferred to the PVDF membrane for electrotransfer. The transformed membrane was blocked with 5% skimmed milk at room temperature for 1 h. The primary antibodies including cleaved-caspase-3, cleaved-caspase-9, cleaved-caspase-1, cleaved-caspase-12, GRP78, CHOP, IL-1β, N-GSDMD, TGFB1, and β-actin (1:1000, Abcam, Cambridge, Britain) were diluted and incubated overnight at 4°C, and then the secondary antibody (1:3000, Abcam, Cambridge, Britain) was diluted and incubated for 30 min at room temperature, and washed 3 times with TBST. Proteins were detected by the chemiluminescence method, and the relative protein expression of each group was calculated by the ImageJ analysis software (Thermo Fisher Scientific Inc., USA). Analysis on each protein sample was repeated 3 times [19 (link)].
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