Western Blot Analysis of Insulin Signaling

Western blot was performed as previously described (Zhang et al., 2019a (link)). Briefly, HepG2 cells were seeded in 60 mm culture dishes (5 × 10⁵ cells) for 24 h. After the treatments for 24 h followed by insulin (100 nM) incubation for 20 min, cells were washed with cold PBS, and then lysed with cold Radio Immunoprecipitation Assay (RIPA) protein extraction buffer containing 1% protease and phosphatase inhibitors (Beyotime biotechnology, Beijing, China) for 30 min on ice. An aliquot of 20 μg of the supernatant protein from each sample was heated with 4 × sodium dodecyl sulfate (SDS) sample buffer at 95 °C for 8 min, and separated electrophoretically on a 10% SDS–polyacrylamide gel. Subsequently, proteins were transferred onto PMSF membranes for 3 h and blocked for 1 h. PMSF membranes were then exposed to indicate primary antibodies in blocking buffer at 1:1,000 dilutions overnight at 4°C. The membranes were then incubated with the anti-rabbit IgG secondary antibody or and anti-mouse IgG HRP-linked antibody at 1:2,000 dilutions for 1 h. Visualization was performed using Tanon 5200 Multi chemiluminescent imaging system (Tanon Science & Technology Co., Ltd., Shanghai, China) with enhanced-chemiluminescence substrate, and the blots were analyzed using Image J software. Protein levels were corrected with values determined on β-actin blots. Three replicates were used.