Western Blot Analysis of Muscle Proteins

Protein extraction and western blot analysis were performed as reported from our previous publication [12 (link)]. The antibodies used were as follows: mouse anti-Pax7 (1 : 500, DSHB AB_528428), rabbit anti-MyoD (1 : 500, Santa Cruz sc-760), mouse anti-MyHC (1 : 500, DSHB MF20), rabbit anti-Tom20 (1 : 1000, Cell Signaling 42406), mouse anti-Sirt1 (1 : 1000, Cell Signaling 8469), rabbit anti-acH3 (1 : 1000, Cell Signaling 9649), rabbit anti-phospho AMPK (Thr172) antibody (1 : 1000, Cell Signaling 2535), rabbit anti-AMPK (1 : 1000, Cell Signaling 2603), rabbit anti-phospho P70S6K (Thr421/Ser424) (1 : 1000, Cell Signaling 9204), rabbit anti-P70S6K (1 : 1000, Cell Signaling 9202), rabbit anti-phospho RPS6 (Ser240/244) antibody (1 : 1000, Cell Signaling 2215), rabbit anti-RPS6 (1 : 1000, Cell Signaling 2217), and rabbit anti-tubulin antibody (1 : 500, Santa Cruz sc-9104). Densitometric analysis was performed using ImageQuant. Normalization of phosphorylated and total proteins was performed by using tubulin or vinculin. Finally, the ratio between the phosphorylated and total protein was indicated.