Ab124937

We use RIPA lysis buffer containing 1 mM PMSF to lyse C2C12 cell or muscles. For the nuclear or cytoplasmic protein extraction, proteins were isolated according to the procedure of the nuclear extraction kit (Solarbio, SN0020). Protein concentration was determined using a BCA protein assays kit. After sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis gels, primary antibodies were used, including rabbit anti‐β‐tubulin (bs‐1482M, 1:5,000, Bioss), rabbit anti‐SUNCR1 (NBP1‐00861, 1:1,000, Novus), mouse anti‐MyHC I (ab11083, 1:1,000; Abcam), rabbit anti‐MyHC IIa (ab124937, 1:1,000, Abcam), goat anti‐MyHC IIb (sc‐168672, 1:500; Santa Cruz), mouse anti‐PGC‐1α (ST1202, 1:1,000, Millipore), rabbit anti‐histone (4499S, 1:2,000; CST), mouse anti‐NFAT (sc‐7294, 1:500; Santa Cruz), rabbit anti‐NRF‐1 (#12381s, 1:2,000, CST), rabbit anti‐calcineurin (#2614s, 1:2,000; CST), rabbit anti‐Myoglobin (ab77232, 1:1,000, Abcam), and rabbit anti‐MEF2A (#97365, 1:2,000; CST). Protein expression levels were determined using MetaMorph software (ImageJ, National Institutes of Health, USA).

Frozen GAS and C2C12 cells were homogenized in RIPA buffer (R0010, Solarbio) containing 1 mM PMSF (P0010, Solarbio), and protease inhibitor cocktail (539133, Merck, Rahway, NJ, USA). The protein concentration was measured using a BCA protein assay kit (P0012, Beyotime, Shanghai, China). Equivalent proteins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinyl difluoride (PVDF) membranes (ISEQ00010, Merck). After blocking the membrane with 5% non-fat dry milk for 1.5 h, primary antibodies were proceeded overnight at 4 °C and included rabbit anti-Fgf21 (ab171941, Abcam), rabbit anti-MyHC I (22280-1-AP, Proteintech), rabbit anti-MyHC IIa (ab124937, Abcam), rabbit anti-MyHC IIb (20140-1-AP, Proteintech), and mouse anti-MyHC IIx (67299-1-Ig, Proteintech), rabbit anti-TGF-β1 (bs-0086R, Bioss, Beijing, China), rabbit anti-Smad2/3 (PA5-99539, Invitrogen), rabbit anti-p-p38 MAPK (8690, CST, MA, USA), mouse anti-Tubulin (T6199, Sigma-Aldrich, MO, USA), and mouse anti-β-actin (4967, CST). HRP-conjugated goat anti-mouse (CW0102S, CWBIO, Taizhou, China) or anti-rabbit (CW0156S, CWBIO) secondary antibodies were incubated at room temperature for 1 h. ECL (PEOO10, Solarbio) was used for enhanced chemiluminescence detection, according to the manufacturer’s instructions.