Western Blot Analysis of Protein Expression

Western blot analysis of protein expression was performed as in our previously described protocol (36 (link)). Following quantification of protein lysates from BMMSCs, the samples (50 µg) were loaded on an SDS-PAGE gel and electroblotted onto polyvinylidene difluoride (PVDF) membranes with use of an electrophoretic transfer apparatus (Bio-Rad Laboratories) overnight. PVDF membranes were blocked with 5% fat-free dry milk solution and incubated with primary antibodies (1:1,000 dilution, anti-FNDC5/Irisin antibody, no. ab131390; Abcam), anti-NLRP3 antibody (ab263899; Abcam); anti–caspase-1 (sc-56036; Santa Cruz Biotechnology); anti–cleaved GSDMD (50928; Cell Signaling Technology); anti–caspase-3 (sc-56053; Santa Cruz Biotechnology); anti-RUNX2 (sc-390351; Santa Cruz Biotechnology); anti-OCN (ab93876; Abcam), and anti-GAPDH 6C5 (CB1001; Merck Millipore) for 2 h at room temperature. The PVDF membranes were then incubated with secondary antibody conjugated with horseradish peroxidase (1:5,000) at room temperature for 1 h, followed by chemiluminescence detection (Clarity Max Western ECL Substrate; Bio-Rad Laboratories). The protein bands were quantitatively analyzed with an Image Lab Software system (Bio-Rad Laboratories).