Anti myogenin

The presence of MyoD and myogenin proteins in C2C12 cell after venom incubation and laser irradiation was detected by Western blotting. Briefly, C2C12 cells were divided into tubes (1x10⁴ cells), received venom or medium (control) and centrifuged for the formation of cell pellet. They were then irradiated in a punctual manner at the bottom of the tube. Subsequently, the cells were placed on 24 well plates and incubated for 15, 30 and 60 minutes. After 3 days, cells were washed with cold phosphate buffer saline (PBS) and directly lysed with Laemmli sample buffer. Then, the samples were run on 10% SDS-PAGE, followed by transfer to a nitrocellulose membrane (Bio-Rad–USA). The membrane was blocked for 1 h at room temperature with 5% non-fat milk, 0.05% Tween-20 in phosphate buffer saline. Then the membrane was incubated with antibodies anti-MyoD (1:500, Abcam®, USA), anti-Myogenin (1:2000, Abcam®, USA), or anti-β-actin, (1:1000, Santa Cruz Biotechnology Inc.) overnight at 4°C. The membrane was washed and incubated with horseradish-peroxidase-conjugated secondary antibody at room temperature for 2 h and the immunoreactive bands were visualized using a chemiluminiscense detection system (Imuno-Star, Bio-Rad). Relative band intensity was quantified using ImageJ software after normalizing with β-actin band intensity.

Cells were fixed with 4% paraformaldehyde in PBS 5 days after GDF‐8 treatment. Fixed cells were blocked in PBS‐T containing 2% BSA for 1 h, and then incubated overnight at 4 °C with anti‐tenomodulin (Santa Cruz Biotechnology, Dallas, TX, USA) and anti‐myogenin (Abcam) antibodies. Subsequently, cells were stained with Alexa Fluor 488‐ and 588‐conjugated goat anti‐(rabbit IgG) secondary antibody (Thermo Fisher Scientific) at room temperature for 1 h. Cells were then mounted using the Vectashield HardSet Mounting Medium with 4′,6‐diamidino‐2‐phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA).