Myog f5d

Protein extraction and western blot analysis were conducted as previously described [52 (link)]. Briefly, total protein was isolated from cells or tissues using RIPA buffer containing 50mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 0.5% sodium Deoxycholate and 0.1% SDS. Protein concentrations were determined using the Pierce BCA Protein Assay Reagent (Pierce Biotechnology). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation) and after blocking in 5% fat-free milk for 1 hour at RT, the filters were incubated with first antibodies in 5% milk overnight at 4°C. The antibodies used for this work were: Desimin, pS6, S6, phospho-GSK3β (Ser9), β-catenin (Cell Signaling), GSK3β (BD Bioscience), Pax7, MyoG (F5D) (Developmental Studies Hybridoma Bank), rabbit anti -Ki67 and active Caspase 3 (Abcam), mouse anti-Ki67, laminin, MyoD, pAMPK, AMPK, and GAPDH (Santa Cruz Biotechnology). Secondary antibodies (anti-rabbit IgG or anti-mouse IgG, Jackson ImmunoResearch) were diluted 8,000-fold. Immunodetection was performed using enhanced chemiluminescence (ECL) with Western blotting substrate (Pierce Biotechnology) and detected with a Gel Logic 2200 imaging system (Carestream).

Protein extraction and western blot were conducted as previously described (Shan et al., 2014 (link)). Briefly, total protein was isolated from cells using RIPA buffer. Protein concentrations were determined using Pierce BCA Protein Assay Reagent (Pierce Biotechnology). Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore Corporation) and detect with specific antibodies. Phospho-S6 (p-S6) and S6 antibodies were from Cell signaling, MF20, Pax7 and MyoG (F5D) were from Developmental Studies Hybridoma Bank (DSHB), all other antibodies (MyoD, pAMPK, AMPK, and GAPDH) were from Santa Cruz Biotechnology (Santa Cruz). Secondary antibodies (anti-rabbit IgG or anti-mouse IgG, Jackson ImmunoResearch) were diluted 8,000-fold. Immunodetection was performed using enhanced chemiluminescence (ECL) Western blotting substrate (Pierce Biotechnology) and detected with a Gel Logic 2200 imaging system (Carestream).