Mouse anti mhc mf 20

C2C12 myoblast cells were washed with PBS in a laminar flow hood and fixed with 4% paraformaldehyde for 10 min at RT in a chemical hood. Cells were permeabilized with 0.25% Triton-X100 in PBS containing 2% bovine serum albumin (Sigma) for 1 hr at RT. Cells were incubated with primary antibody at 4°C overnight, then incubated with secondary antibody at RT for 1 hr. Primary antibodies included mouse anti-Hnrnpa2b1 (ab6102, Abcam) at 1:200, mouse-anti-myogenin (ab82843, Abcam), and a mouse anti-MHC MF-20 (Developmental Studies Hybridoma Bank, University of Iowa) at undiluted, ‘neat’ concentration. Alexa Fluor secondary antibodies (Molecular Probes) were used at a 1:1,000 dilution.

Immunostaining was performed on 4% paraformaldehyde fixed cells, treated with 0.3% Triton X and blocked with 3% BSA (all from Sigma). Primary and secondary fluorochrome conjugated antibodies were diluted in 3% BSA and incubated overnight at 4 °C or for 1 hour at room temperature.
Western blots were performed on proteins separated on 8% SDS-PAGE gels and transferred to PVDF membrane. Primary and secondary HRP conjugated antibodies were diluted in 5% skim milk in TBST and incubated overnight at 4 °C or 1 hour at room temperature. HRP signal was visualized using Pierce ECL western blotting substrate (Thermo Scientific).
The following antibodies were used: mouse anti-MHC (MF20, 1:20, Developmental Studies Hybridoma Bank), rabbit anti-MyoD (1:200, Santa Cruz), rabbit anti-DUX4 (RD2-47c, 1:50, R&D Systems), Alexa fluor 488 Goat Anti-Mouse, Alexa fluor 555 Goat Anti-Rabbit (1:500, Invitrogen), Flag M2 (Sigma), GAPDH-HRP (1:5000, GenScript), 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen). Full western blot images are presented in the Supplementary Figs S1 and S2.