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Muscles from humans and mice were lysed with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 5 mM Na3VO4, 20 mM NaF, 10 mM sodium pyrophosphate, protease inhibitor cocktail [Roche, #04693132001], 1 mM phenylmethylsulfonyl fluoride (PMSF) [Generay Biotech, #0754-PMSF], 1% Triton-X, and 0.1% SDS [Biosesang, #S2003]) and homogenized using a homogenizer. After centrifugation (15,000 × g for 15 min at 4 °C) to remove debris, total protein concentration of the supernatants was determined with the Pierce BCA protein assay kit (Thermo Scientific, #K1672).
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