Phosphorylated p65 p p65

Cells were lysed by using RIPA Lysis buffer (Sigma Aldrich) containing Protease Inhibitor Cocktail (1 mL/10 mL lysates, Sigma Aldrich) and PhosSTOP™ (1 tablet per 10 mL lysate, Roche). The lysates were incubated for 20 min at 4°C with shaking, followed by centrifuged (130000g, 10 min, 4°C) and the supernatant was used for automated capillary-based immunoassay on a Wes Simple Western System (ProteinSimple, Bio-Techne, Minneapolis, USA). Primary antibodies were used as followed Actin-β (dilution 1:100; catalog no. A5441, Sigma Aldrich), p65 (1:50; 8242, Cell Signaling Technology, Danvers, MA, USA), phosphorylated-p65 (p-p65) (1:50; 3033, Cell Signaling Technology), STAT6 (1:50; 5397, Cell Signaling Technology), p-STAT6 (1:50; 56554, Cell Signaling Technology), STAT1 (1:50; 9172, Cell Signaling Technology) and p-STAT1 (1:50; 7649, Cell Signaling Technology). These antibodies were diluted with antibody diluent 2 (ProteinSimple). Secondary antibody was horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (ProteinSimple) or anti-mouse IgG antibody (ProteinSimple) and Lumino-S/peroxidase (ProteinSimple) was used as substrates for HRP.

Proteins extracted from heart tissue and cardiomyocytes. Protein concentration was detected by the BCA Protein assay kit (PC0020, Solarbio, Beijing, China). Equal amounts of protein were added and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The membranes were then blocked with 5% fat-free milk solution at room temperature for 1 h. Then, the membranes were incubated with primary antibodies against GSDMD (1:1,000, ab219800, Abcam), NLRP3 (1:1,000, ab263899, Abcam), caspase-1 (1:200, sc-56036, Santa Cruz Biotechnology), NF-κB P65 (1:1,000, #8242, Cell Signaling Technology), phosphorylated P65 (p-P65) (1:1,000, #3033, Cell Signaling Technology) and GAPDH (1:1,000, #5174, Cell Signaling Technology) at 4°C overnight, followed by secondary antibody for 1 h at room temperature. After washing three times, enhanced chemiluminescence (ECL) reagent was added to the membrane. The signal was detected using an ECL system.