Protein Extraction and Western Blot Analysis

Proteins were extracted by using Ripa buffer (Thermo Fisher Scientific, Waltham, MA,USA) and concentrations were calculated with BCA assay (Thermo Scientific, Waltham, MA, USA) as previously reported [22 ]. Protein samples were diluted in Laemmli’s sample buffer (Biorad, CA, US), heated for 5 min at 95 ◦C, and separated by sodium dodecyl sulfate − polyacrylamide gel electrophoresis (SDS PAGE), followed by transfer onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Waltham, MA, USA). PVDF membranes were probed with the primary (antiMyoD (MA1-41017; Thermo Scientific, Waltham, MA, USA), β ACTIN (BA3R; Thermo Scientific, Waltham, MA, USA); and secondary antibodies ( Anti-mouse (Cell Signaling, 7076). Signals were detected with a chemiluminescence reagent (ECL, Millipore, Burlington, MA, USA) and Images were recorded using a LAS4000 Digital Image Scanning System (GE Healthcare, Little Chalfont, UK). Densitometric analysis was performed as we previously reported [24 (link)].