Bs 0295g

Western blots were performed on samples of pectoral major muscle, as previously described (Frolova et al. 2009 (link)). In total, 50 mg of muscle were grinded with liquid nitrogen and then the powder was lysed in 1 ml lysis buffer, 50 mM Tris HCl (pH 7.5), 150 mM NaCl, 5 mM “ethylenediaminetetraacetic acid, 0.5% Nonidet P-40, and completeMini protease inhibitor cocktail (BC3640; Solarbio). The samples were placed on ice for 4 min and then centrifugated at 12,000 rpm for 15 min at 4 °C. In total, 350 μl of the supernatant were transferred to a 2-ml auto-sampler vial and were diluted by 3-fold of loading buffer. Protein concentration was quantified using the BCA Protein Assay Kit (PC0020; Solarbio). Supernatant was then separated using Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for 1 h at 140 V and transferred to PVDF for 2 h at 90 V. Nonspecific antibody bind in was blocked in 5% nonfat dry milk powder in 1 Tris-buffered saline for 1 h. Blots were incubated in 1% nonfat dry milk powder in Tris-buffered saline with 0.05% Tween 20 overnight at 4 °C with the following antibodies: rabbit anti-GLUT12 (bs-2540R; 1:1,000; Bioss), and mouse anti-β-actin (bsm-33036M; 1:1,000; Bioss). Blots were incubated with goat anti-rabbit (bs-0295G; 1:3,000; Bioss) for 1 h at room temperature. Blots were detected using protein detection reagents (AY0371; Solarbio).