Western Blotting Protocol for Muscle Proteins

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.