Quantitative Protein Expression Analysis

Western blot analysis was performed as previously reported [40 (link)]. The cultured cells were lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology, China) supplemented with 1 mM PMSF (Invitrogen), and then, the protein concentration was determined using a BCA protein assay kit (Thermo Fisher). Equal amounts of proteins were separated by 10% SDS-PAGE electrophoresis and transferred to a 0.22-μm PVDF membrane (Millipore Corporation, Billerica, MA, USA). The membrane was blocked with 5% BSA for 1 h in room temperature and was then incubated with the following primary antibodies: anti-myoG (1:1000, Abcam), anti-myoD (1:1000, Abcam), anti-phospho Akt (1:1000, Abcam), anti-Akt (1:1000, Abcam), and anti-β-actin (1:3000, Abcam) at 4 °C overnight. Then, the membrane was incubated with an anti-rabbit (1:5000) or anti-mouse (1:5000) fluorescein-conjugated secondary antibody (Abcam). The immune bands were visualized by Odyssey V3.0 image scanning. All of the procedures were performed three times, and the bands were selected from individual results. Quantitative analysis was performed using ImageJ software; the normalized greyscale was calculated by dividing the individual greyscale of each marker to the β-actin.