Anti myod1

The total proteins were extracted from different treatment groups using lysis buffer (Solarbio) and centrifuged at 12,000 rpm for 30 min at 4°C. The protein concentration was measured using a BCA protein assay kit (BestBio, Shanghai, China). Additionally, the protein was separated by 10% SDS-PAGE and transferred to a PVDF membrane (Millipore Corporation, Billerica, MA). The membrane was blocked with closed solution (Beyotime, Shanghai, China) and then incubated with different primary antibodies, respectively. The following primary antibodies were used: anti-MYOG (Biorbyt, Cambridge, UK; 1:1,000) and anti-MYOD1 (Santa Cruz Biotechnology, Dallas, TX, 1:1,000), anti-MTOR, anti-p-MTOR, anti-P70S6K, anti-P-P70S6K, and anti-β-tubulin (Zenbio, Chengdu, China, 1:1,000) for 12 h. The secondary antibodies used were: mouse anti-rabbit horseradish peroxidase (HRP), goat anti-mouse HRP (Zenbio, 1:1,000). Finally, protein stripes signals were promoted by the ECL kit (Beyotime) and detected by the ImageJ (NIH Image J System, Bethesda, MD). β-Tubulin was used as the internal reference.