2.4. Western Blot Analysis

Skeletal muscle and liver homogenates in buffer were centrifuged at 20,000× g (4 °C) for 15 min. A protein sample of 20 μg was loaded on a 10% SDS-PAGE gel and transferred to a nitrocellulose membrane. The isolated proteins were blocked with 5% skimmed milk and 0.1% Tween20 in Tris buffer for 60 min. Blocked membranes were incubated with primary antibodies against IRS-1ser³⁰⁷, IRS-1tyr⁶¹², p85-PI3K, AKTser⁴⁷³, PM-GLUT4, FOXO1, G6Pase, PEPCK, GSK3β, and GS for 60 min (Abcam, Cambridge, UK). The membrane was washed, the secondary antibody was incubated for 60 min, and each antigen-antibody complex was visualized using a Western blotting detection reagent. Chemiluminescence was detected by a LAS-1000 Analyzer (Fujifilm, Tokyo, Japan), and band density was measured using an Image Analyzer.