Anti myog

The cells were fixed in 4% paraformaldehyde for 15 min and then permeabilized in 0.3% Triton X-100 for 15 min. Subsequently, the cells were blocked with blocking solution (3% bovine serum albumin (BSA), 0.3% TritonX-100, 10% FBS complemented with PBS) for 2 h. Then, Anti-MyHC (1:1000; Millipore, Billerica, MA, USA) or anti-MyoG (1:500; Abclonal, Wuhan, China, Cat#A17427) were added and the solution was incubated overnight at 4 °C. After that, the cells were stained with CoraLite594-conjugated Goat Anti-Rabbit IgG (H + L) (Proteintech, Wuhan, China, Cat#SA00013-4) for 1 h. The cell nuclei were stained using DAPI (Servicebio, Wuhan, China, Cat#G1012-10ML) solution in darkness for 10 min. Images from three randomly selected fields were obtained with a Leica SP8 confocal microscope and processed with Image J software (version 1.53e).

The protein expression levels of the myogenin (MyoG) gene, myogenic differentiation (MyoD), myosin heavy chain (MyHC), and CDC23 in the PSCs were detected by performing immunoblotting. Transfected cells were lysed in RIPA buffer with 1% PMSF and the protein was loaded onto an SDS-PAGE gel and transferred onto a PVDF membrane. Non-specific binding was blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 for 2 h. Then, the proteins were incubated with anti-MyoG (1:1000, Abclonal, Wuhan, China, Cat#A17427), anti-MyoD (1:1000, Proteintech, Wuhan, China, Cat#18943-1-AP), anti-myosin heavy chain (MyHC; 1:3000, Millipore, Darmstadt, Germany, Cat#05-716), CDC23 antibody (1:1000, Abclonal, Wuhan, China, Cat#A6025), and anti-β-tubulin (1:3000, Proteintech, Wuhan, China, Cat#10068-1-AP) at 4 °C overnight. The blots were subsequently incubated with HRP-conjugated secondary antibodies (1:4000), including HRP-labeled goat anti-mouse IgG (Servicebio, Wuhan, China, Cat#GB23301), and HRP-labeled goat anti-rabbit IgG (Servicebio, Wuhan, China, Cat#GB23303). ECL substrates were used to visualize the signals (Beyotime, Shanghai, China, Cat#P0018A). Image J software (version 1.53e) was used to conduct a quantitative analysis of the Western blotting results, according to the gray value of the strip.