Enhanced bca protein detection kit

The protein sample was extracted by RIPA protein extraction reagent (Beyotime, Beijing, China), and the protein concentration was determined by the enhanced BCA protein detection kit (Beyotime, Beijing, China). Equal amounts of protein were separated by SDS–PAGE gel electrophoresis and then transferred to PVDF membranes (0.2 μm; Bio-Rad) overnight. After blocking with 5% skimmed milk for 1 h, the membranes were incubated with primary antibody RUNX2 (CST, 8486s) at 4°C overnight. The membranes were incubated with secondary antibodies at room temperature for 1 h after washing three times with 1×TBST. Finally, target protein was visualized by ECL Western Detection Kit (Thermo Fisher Scientific, United States) and quantified by ImageJ software.

Muscle proteins were extracted via RIPA buffer containing 1% phenylmethanesulfonyl fluoride as well as protease and phosphatase inhibitor cocktails from Roche (04693124001, 4906837001, Basel, Switzerland). Protein concentration was determined using the enhanced BCA protein detection kit (#P0010, Beyotime, Shanghai, China). The detailed process of the Western blot was described in our previous report [23 (link)]. In brief, the protein samples were mixed with the loading buffer and boiled for 5 min. Then, the obtained samples were loaded and separated using 10% or 12% SDS–PAGE gels. When the electrophoresis was finished, the gels were transferred to polyvinylidene fluoride membranes. After that, the membranes were blocked with 5% nonfat milk dissolved in TBS-T buffer, and then incubated with the indicated primary antibodies (~1:1000) overnight. The membranes were then thoroughly washed and incubated with the corresponding horseradish peroxidase-labeled secondary antibodies (~1:10,000). To visualize the blots, the membranes were reacted with the chemiluminescent substrate and then subjected to the ChemiDoc™ XRS+ Gel Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) for image acquisition.