Insulin receptor β

RIPA lysis buffer (Beyotime) was adopted to acquire protein sample. Then the equal amount protein samples were loaded on 8–12% polyacrylamide gel for electrophoresis separation, followed by blotting onto a polyvinylidene difluoride membrane (Thermo Fisher Scientific, USA). Blocking was performed by incubation in 5% bovine serum albumin (BSA, Biosharp, Guangzhou, China) for 1 h. Then the membranes were probed with primary antibodies SREBP1 (1:1000, Proteintech, China), CYP17 (1:500, Proteintech), CYP19 (1:500, Proteintech), AMPKα (1:1000, Cell Signaling Technology, USA), p-AMPKα (1:1000, Cell Signaling Technology), p-AKT (1:2000, Cell Signaling Technology), AKT (1:1000, Cell Signaling Technology), p-IR (1:1000, Cell Signaling Technology), insulin receptor β (IR, 1:1000, Cell Signaling Technology), cytochrome C (1:1000, Abclonal, China), β-actin (1:2000, Proteintech) at 4 ℃ overnight. Goat anti-rabbit or anti-mouse IgG (1:10000, Proteintech) was used as secondary antibody. The antigen–antibody complexes were visualized with ECL Plus reagent (7 sea biotech, Shanghai, China).

Proteins were extracted from the heart tissue in a 1x RIPA buffer (Melford, Ipswich, UK). The protein concentration was determined with Bradford assay. Total protein homogenates 20 μg/30 μl were denatured, separated on sodium dodecyl sulfate polyacrylamide electrophoresis gels, and transferred to a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was blocked with 2.5 % BSA in Tris- Buffered Saline Tween 20 for 1 h, before being incubated overnight at 4 °C with CD36 (1:1000, Cell Signalling Technology, Cambridge, UK), GLUT-4 (1:100, Santa Cruz, Biotechnology, Heidelberg, Germany), insulin receptor-β (1:1000, Cell Signalling Technology, Cambridge, UK), phosphorylated-PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), PI3K (1:1000, Cell Signalling Technology, Cambridge, UK), AKT, or phosphorylated-AKT (1:500, Abcam, Cambridge, UK). After washing the blots to remove excessive primary antibody binding, the blots were incubated for 1 h with a horseradish peroxydase conjugated secondary antibody at room temperature. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), housekeeping protein, was used for loading control and protein normalization. The immunoreactive protein bands were developed using the Enhanced Chemiluminescence system (PerkinElmer, Rodgau-Juegesheim, Germany). The intensity of the immunoblot bands was detected with Chemismart 5100.