Wl02785

Total bovine primary myoblasts proteins were extracted from different treatment groups using RIPA lysis buffer containing 1mM PMSF (Solarbio; Beijing, China). The protein extract was then boiled for 10 min in a loading buffer of 5× SDS-PAGE. The protein was then separated by SDS-PAGE and transferred to a 0.2 μm polyvinylidene fluoride membranes (PVDF). Then incubated for 2 h at room temperature with 5% skim milk in Tris Buffered Saline, with Tween (TBST) to block the PVDF membrane. Subsequently, in 4 °C overnight incubation with the primary anti-MYOD specific antibodies (Dilution 1:1000; ab16148; Abcam, Cambridge, UK), anti-MYOG (1;1000; ab124800; Abcam), anti-MYH2 (Dilution 1:1000; WL02785; Wanleibio, Shenyang, China), anti-CDK2 (Dilution 1:1000; WL02028; Wanleibio), anti-PCNA (Dilution 1:500; WL03069; Wanleibio), anti-CyclinD1 (Dilution 1:1000; WL01435a; Wanleibio), anti-β-actin (1:1000; KM9001T, SungeneBiotech, Tianjin, China). After that, the PVDF membranes were washed with TBST buffer and then incubated at room temperature for 2 h with the secondary antibody. The secondary antibodies were: goat anti-mouse IgG HRP (M21001S, Abmart, Shanghai, China), goat anti-rabbit IgG (H + L)-HRP (BA1054, Boster, Wuhan, China). Finally, ECL luminous liquid (DiNing, Beijing, China) was used to detect the protein bands.