P irs 1

eWAT or THP-1 macrophages were harvested using RIPA lysis buffer (Beyotime, China), and immunoblots were performed as previously described [12 (link)]. Briefly, the samples were separated on 8% SDS polyacrylamide gels and then transferred to the nitrocellulose membrane. The membrane was incubated with the corresponding primary antibodies overnight, followed by appropriate horseradish peroxidase-conjugated secondary antibodies for 1.5 h. The bands were detected by an enhanced chemiluminescence (ECL) detection system (Beyotime, China). The images of the chemiluminescence were captured with an imaging system (Bio-Rad, Hercules, CA, USA). The optical densities were normalized to those of β-actin, and the fold difference for each target protein was calculated as the ratio of the target protein expression/β-actin expression (ImageJ 1.42q software, National Institutes of Health, USA). The primary antibodies are shown as follows: ClC-3 (1:200, Alomone Labs, Israel), TLR-4 (1:500, Santa Cruz Biotechnology, USA), NF-κB p65 (1:1000, Cell Signaling Technology, USA), p-NF-κB p65 (1:1000, Cell Signaling Technology, USA), IRS-1 (1:500, Santa Cruz Biotechnology, USA), p-IRS-1 (1:500, Santa Cruz Biotechnology, USA), and β-actin (1:2000, Cell Signaling Technology, USA).