Western Blot Analysis of IRS1 and GAPDH

As described by Feng et al [33 (link)]., cells were collected and immersed in RIPA lysis buffer (Solarbio, Beijing, China) and supplemented with phenylmethanesulfonyl fluoride. A BCA Protein Assay Kit was used to determine the protein concentration. Equal amounts of protein were subjected to 10% SDS-PAGE and then transferred to a polyacrylamide difluoride membrane. After blocking with 5% nonfat milk for 2 h and subsequent incubation with primary antibodies at 4°C overnight, membranes were probed with HRP-labeled secondary antibody (ab150077; Abcam) at room temperature for 2 h. The immunoreactive bands were detected with an enhanced chemiluminescence (ECL) system (Pierce). The following primary antibodies were used in this study: anti-IRS1 (ab40777; Abcam) and anti-GAPDH (ab181602; Abcam).