Bicinchoninic acid protein concentration quantification kit

Total protein of rat liver tissue in each group was extracted using radioimmunoprecipitation assay lysis buffer. The protein concentration was determined using a bicinchoninic acid protein concentration quantification kit (Beyotime Biotechnology, Shanghai, China). Denatured proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membranes were blocked with 5% nonfat dry milk in tris-buffered saline containing 0.05% Tween 20 or 1–3% bovine serum albumin and probed with primary antibodies overnight at 4°C. The primary antibodies used included IRS-1, Akt (1 : 1000 dilution, Proteintech Inc., Wuhan, China), p-IRS-1 (1 : 1000 dilution, Cell Signaling, USA), p-Akt^Ser473 (1 : 2000 dilution, Abcam, USA), and β-actin (1 : 200 dilution, Boster Biological Technology Co. Ltd., Wuhan, China). Following washing, the membranes were incubated with goat horseradish peroxidase-conjugated secondary antibodies (1 : 50,000 dilution, Boster Biological Technology Co. Ltd., Wuhan, China). The reactions were detected by using an Enhanced Chemiluminescence Western Blot detection kit (Thermo Fisher, USA), and the detected bands were visualized via exposure to an X-ray beam in a dark room.

Total protein of the cell lysate in each group was extracted using NP-40 lysis buffer (Beyotime Biotechnology, Shanghai, China). The protein concentration was determined using a bicinchoninic acid protein concentration quantification kit (Beyotime Biotechnology). Denatured proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. The membrane was blocked with 5% non-fat dry milk in Tris-buffered saline containing 0.05% Tween 20 and probed with primary antibodies overnight at 4°C. Following washing, the membrane was incubated with goat horseradish peroxidase-conjugated secondary antibodies at room temperature for 2 h (1:2,000 dilution, Santa Cruz Biotechnology, Santa Cruz, CA, USA). The reactions were detected by using Enhanced Chemiluminescence Western Blotting Substrate (Pierce, ThermoFisher Scientific, Shanghai, China). The detected bands were visualized via exposure to an x-ray beam in a dark room and semi-quantified via gray-scale analysis.