Bca assay

Mstn protein in rabbit skeletal muscle was detected by Western bloting analysis. The proteins were prepared using radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Haimen, China) and the concentrations were determined using the bicinchoninic acid (BCA) assay (Ding Guo, Nanjing, China). Then, denaturation was performed in sodium dodecyl sulfate (SDS) gel-loading buffer at 100 °C for 10 min. Total protein (40 μg per sample) were separated by 12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore; Billerica, USA). After incubation in blocking buffer (5% BSA in Tris-buffered saline containing 0.1% Tween 20) for 1 h at RT, the membrane was incubated overnight at 4 °C with a mouse anti-Mstn primary antibody (Sigma, SAB5300419, 1:1,000 dilution) and mouse anti-β-actin primary antibody (Santa Cruz Biotechnology, SC-47778, 1:1,500 dilution). After washing, the membrane was incubated with a goat anti-mouse IgG (H + L) secondary antibody (Thermo Pierce, 31160, 1:5,000 dilution) for 1 h at RT. After washing, the signal was detected using an ECL western blotting detection system (Fujifilm, Tokyo, Japan), and the chemiluminescence intensity of each protein band was quantified using ImageJ software.

Cells were washed thrice with cold PBS and lysated in 100 μL RIPA lysis reagent (Ding Guo Biotechnology Co., Ltd, China) added 2 μL PMSF and 2 μL phosphatase inhibitor (Ding Guo Biotechnology Co., Ltd, China) for 30 min. Cells debris was removed by centrifugation at 14,000×g for 20 min at 4 °C. Protein concentration was measured by BCA assay (Ding Guo Biotechnology Co., Ltd, China). Clarified proteins lysated from each experimental condition (50 μg) were boiled for 5 min. Protein samples were subjected to 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membrane (GE Healthcare life science, USA). After blocked with 5% non-fat milk in 1× TBST containing 0.05% Tween-20 for 2 h at room temperature, the membranes were incubated with specific primary antibodies overnight at 4 °C. Anti-mouse IgG and anti-rabbit IgG were used as secondary antibodies (Biosharp, China). Detection of specific proteins was performed by enhanced chemiluminescence reagent (ECL, Advansta, USA). The antibodies were as follows: anti-AMPKα (ref #ab32047, 1:3000, Abcam), Anti-phospho-specific (Thr172) AMPKα (ref #ab133448, 1:5000, Abcam), Anti-p21 (ref #ab109520, 1:2000, Abcam) and Anti-GAPDH (ref #10494-1, 1:5000, Bioworld).