Sc 4777

Cells and muscle tissue were lysed in RIPA buffer (Beyotime Biotechnology, Jiangsu, China) according to the manufacturer’s instructions. Protein lysates were heated at 95°C for 5 min in 5 × SDS sample buffer, then separated by 10% SDS polyacrylamide gel electrophoresis (30 μg each lane), followed by transfer to a polyvinylidene fluoride membrane (Millipore, Burlington, MA, United States) using a Mini Trans-Blot Cell system (Bio-Rad). The membrane was blocked with 5% non-fat milk for 3 h. The primary antibodies were incubated overnight at 4°C. The membranes were washed and incubated with secondary antibodies for 1 h at 37°C followed by visualization by enhanced chemiluminescence (Bio-Rad). Primary antibodies specific for EZH2 (ab3748, 1:1,000; Abcam, Cambridge, United Kingdom), MyoD (sc-760, 1:1,000; Santa Cruz Biotechnology, Dallas, TX, United States), MyoG (sc-12732, 1:200; Santa Cruz Biotechnology), MyHC (sc-376157, 1:3,00; Santa Cruz Biotechnology), Ki67 (ab16667, 1:1,000; Abcam), p21 (sc-6246, 1:1,000; Santa Cruz Biotechnology), and β-actin (sc-4777, 1:1,000; Santa Cruz Biotechnology), along with goat anti-mouse IgG-HRP (sc-2005, 1:3,000; Santa Cruz Biotechnology) and goat anti-rabbit IgG-HRP (sc-2004, 1:3,000; Santa Cruz Biotechnology) secondary antibodies were used to detect protein expression.

Proteins from the cell lysates were prepared in 100 mg/mL RIPA lysis buffer containing 1% (v/v) phenylmethylsulfonyl fluoride (Beyotime, China), then centrifuged at 12,000 g for 10 min at 4 °C. After that, the lysates were heated at 95 °C for 5 min in 5× sodium dodecyl sulfate (SDS) sample buffer. Identical quantities of proteins were separated by 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), and non-specific binding was blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 for 4 h. The membranes were incubated overnight with primary antibodies at 4 °C, washed three times, and then incubated with the secondary antibodies for 1 h at 37 °C. The antibodies for Western blot analysis were obtained from Santa Cruz Biotechnology, including anti-myogenin (sc-12732; 1:200 dilution), anti-MyoD (sc-760; 1:200 dilution), anti-MyHC (sc-376157; 1:1000 dilution), and anti-β-actin (sc-4777; 1:1000 dilution). Changes in protein levels were normalised to the housekeeping protein β-actin for quantitative Western blot analysis using ImageJ software.