Anti myosin heavy chain

Western blotting and antibodies: total proteins of the C2C12 cells were extracted with RIPA buffer supplemented with a protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentration was determined using a bicinchoninic acid (BCA) protein assay kit (Thermo Scientific, Beijing, USA). The primary antibodies used are as follows: β-tubulin (Cat #10094-1-AP, 1:5,000; Proteintech, Wuhan, Hubei, China), myogenin (Cat # ab1835, 1:2000; Abcam, Cambridge, MA, USA), HMGA1 (Cat #ab129153, 1:1,000; Abcam, Cambridge, MA, USA), and DNMT1 (Cat # 24206-1-AP; Proteintech, Wuhan, Hubei, China). Horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000; Bio-Rad, Hercules, CA, USA) were also used.
Cell immunostaining: The C2C12 cells were cultured on glass coverslip in 24-well plates, then fixed with 4% formaldehyde in PBS buffer, permeabilized with 0.5% Triton X-100 in PBS for 10 min, and blocked with 1% bovine serum albumin for 15 min. The primary antibody anti-myosin heavy chain (1:100; Abcam, Cambridge, MA, USA) was used. Indirect immunofluorescence was detected with fluorescein isothiocyanate-conjugated anti-mouse IgG secondary antibodies (Cat #A-11001; Invitrogen, Carlsbad, CA, USA).

The immunostaining of cells was carried out with slight modifications of the previously reported protocols.^{38 (link)} The C2C12 cells were grown on sterile glass coverslip in 24-well plates, fixed with 4% formaldehyde in PBS for 20 min at room temperature and permeabilized with 0.5% Triton X-100 in ice-cold PBS for 10 min. The pretreated cells were blocked with 1% bovine serum albumin for 15 min and incubated with primary antibodies for 3 h at room temperature or overnight at 4 °C at the following dilutions: anti-Myosin heavy chain (1 : 100; Abcam) and anti-Flag (1 : 25; Proteintech). The indirect immunofluorescence was detected after incubation with fluorescein isothiocyanate-conjugated anti-mouse/rabbit IgG (1 : 500; Abcam). The cell nuclei were stained with 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) for 5 min. After several washes with PBS, the cells were subjected to a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).