Pmsf 1 mmol l

Total protein of the liver was obtained and measured using an radioimmunoprecipitation (RIPA) buffer including PMSF (1 mmol/L) (Beyotime, Shanghai, China) and a BCA assay kit (Beyotime, Shanghai, China), respectively. Protein extracts were mixed with loading buffer and fully denatured in a boiling water bath according to Yang et al. (2021) with minor changes [24 (link)]. Target proteins were subjected to 8–12% SDS-PAGE electrophoresis, and transferred to a polyvinylidene-difluoride (PVDF) membrane (Beyotime, Shanghai, China). Afterwards, PVDF membranes were washed (3 times × 10 min in 1 × PBST), after blocking 2 h in 5% skim milk. After washing 3 times, PVDF membranes were incubated in primary antibody (GAPDH, NLRP3, and caspase-1) for 8–12 h at 4 °C. Related horseradish peroxidase labeled antibody was incubated at 37 °C for 1 h, after washing 3 times in PBS–0.1% Tween 20 (PBST). Protein bands were quantified and were recorded using the Essential V6 imaging platform (UVITEC, Cambridge, England) with the enhanced chemiluminescence (ECL) chemiluminescence kit (Beyotime Biotechnology). The GAPDH protein served as an internal control protein. The protein expression was expressed as the ratio of band intensities of proteins to that of GAPDH.