Gsdmd

H9C2 cardiomyocytes were lysed in iced RIPA buffer (Solarbio, China) containing protease inhibitors for 30 min and centrifuged at 13523 g, 4°C for 10 min to collect the supernatants. The protein concentration was measured by the BCA protein assay kit (Solarbio). Proteins were then separated in sodium dodecyl sulfate (SDS) polyacrylamide gel by electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked with tris-buffered saline with tween 20 (TBST) buffer containing 5% nonfat milk and then incubated with primary antibodies against GAPDH (1:1000, Abcam, UK), Caspase-1 (1:1000, Abcam), NLRP3 (1:1000, Proteintech, China), GSDMD (1:1000, Biorbyt, UK), IL-1β (1:1000, Beyotime, China), and CH25H (1:500, Santa Cruz, USA) overnight at 4°C. After washing with TBST buffer three times, membranes were incubated with secondary antibody (Cell Signaling Technology, USA) for 2 h. The specific protein bands were detected using the Odyssey CLx imaging system (LI-COR, USA).

First, equal amounts of protein were leaded in each lane of a polyacrylamide gel. Next, the proteins were separated by SDS-PAGE and transferred to a PVDF membrane. After incubated for two hours at room temperature, the membrane was blocked with 5% skim milk and sequentially incubated with primary antibodies against NLRP3 (Cell Signaling Technology, Danvers, MA, USA), GSDMD (Biorbyt, St Louis, MO, USA), ASC (Biorbyt), Caspase 1/p20/p10 (Proteintech, Rosemont, USA), MuRF1 (Biorbyt), Atrogin-1 (Biorbyt), Caspase 3 (Proteintech), IL-1β (Biorbyt), IL-18 (Sigma-Aldrich, St. Louis, MO, USA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Bioworld Technology, Minneapolis, MN, USA) overnight at 4 °C. The membrane was then washed and incubated for 2 h at room temperature with horseradish peroxidase-conjugated IgG. Finally, we employed ECL Western blotting detection reagents (Thermo Fisher Scientific) to visualize the protein bands and used Un-Scan-It 6.1 software (Silk Scientific, Orem, UT, USA) to measure the band density.