Ab82843

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.

Frozen gastrocnemius was crushed using a mortar and a pestle cooled with liquid nitrogen. Then, 50 mg of pulverized tissue was homogenized in a buffer containing 15 mM HEPES, 10% glycerol, 0.5% NP-40, 250 mM NaCl, 1 mM EDTA, 1 mM phenylmethanesulfonylfluoride (PMSF), and a phosphatase- and protease-inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined by the BCA. Then, 40 μg of protein was subjected to 10–15% SDS-PAGE and transferred to nitrocellulose membranes. After blocking in 3% skimmed milk (1 h, RT), membranes were incubated overnight at 4 °C with anti-myostatin/GDF8 antibody (0.4 μg/mL; R&D Systems, AF788, Minneapolis, MN, USA), pSTAT3 (2.5 μg/mL; R&D Systems, MAB4607), pSTAT1 (5 μg/mL; Affymetrix eBioscience, 14-9008, San Diego, CA, USA), SOCS1 (1 μg/mL; Abcam, ab9870, Cambridge, UK), SOCS3 (4 μg/mL; Abcam, ab14939), Pax7 (2.8 μg/mL, Developmental Studies Hybridoma Bank, AB 528428, Iowa City, IA, USA), Myogenin (2 μg/mL; Abcam, ab82843), and MyoD1 (1 μg/mL; Abcam, ab16148). Antibody binding was detected with chemiluminescence using secondary antibodies: anti-goat-HRP 1:5000 (Merck Millipore), anti-rat-HRP 1:20,000 (Fisher Scientific) or anti-mouse-HRP 1:5000 (GE Healthcare LifeSciences, Piscataway, NJ, USA. α-tubulin protein levels were employed as the loading control.