Western Blotting of Muscle Differentiation Markers

Cells were lysed in modified RIPA buffer (50mM Tris-HCl pH 7.4, 150mM NaCl, 1% IGEPAL, protease and phosphatase inhibitors) and 10–30μg of total protein was loaded into 10% SDS-PAGE gels and transferred to PVDF membranes for Western blotting with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7, 1:10; mouse monoclonal (F5D) anti-myogenin (Developmental Studies Hybridoma Bank, USA), 1:5; mouse monoclonal anti-tubulin, 1:10000; mouse monoclonal anti-HA HRP conjugated, 1:4000 (Sigma-Aldrich,USA); mouse monoclonal anti-HDAC2 [3F3], 1:5000; rabbit polyclonal ChiP-grade anti-HA tag, 1:10000; rabbit polyclonal anti-Nedd4, 1:10000; rabbit monoclonal anti-GFP E385, 1:5000 (Abcam, UK); mouse monoclonal anti-GAPDH (EMD-Millipore, USA), 1:10000; mouse monoclonal 9B11 myc-tag (Cell Signaling, USA), 1:1000; rabbit polyclonal anti-GST (gift from Dr. María Paz Marzolo, Santiago, Chile), 1:5000 and mouse monoclonal P4D1 anti-ubiquitin (Santa Cruz Biotechnology, USA), 1:500. As secondary antibodies HRP conjugated anti-mouse IgG and anti-rabbit IgG (Cell Signaling, USA) were used at 1:5000. HRP activity was detected using the SuperSignal West Dura Extended Duration Substrate (Thermo-Fisher Scientific, USA). When indicated, lysates/fractions were subjected to co-immunoprecipitation as described [16 (link)].