Mab4470

Total proteins were extracted with Laemmli lysis buffer, sonicated (30 s ON/30 s OFF for 5 min) resolved on 10.5% acrylamide/bis-acrylamide SDS–PAGE gels and transferred to nitrocellulose membranes (Thermo Fisher Scientific) in transfer buffer. Protein transfer was assessed by Ponceau-red staining. Membranes were blocked in Tris-buffered saline pH 7.4 containing 0.1% Tween-20 and 5% milk for 1 h at room temperature. Incubations with primary antibodies were carried out at 4 °C overnight using the manufacturer recommended dilutions in Tris-buffered saline pH 7.4, 0.2% Tween-20. After 1 h incubation with an anti-rabbit or anti-mouse peroxidase-conjugated antibody (Jackson ImmunoResearch) at room temperature, proteins were detected by chemiluminescence using SuperSignal (Thermo Fisher Scientific), with Fusion v15.11 software or with the LI-COR Odyssey FC Imaging System and Image Studio Lite Software. The following antibodies were used: mouse anti-MyHC (R&D Systems, MAB4470), rabbit anti-histone H3 (Abcam, ab1791), rabbit anti-GAPDH (Sigma, G9545), monoclonal anti-Flag M2-Peroxidase (Sigma, A8592), rabbit anti-MCK (Kindly provided by Dr Slimane Ait-si-Ali, raised by Dr H Ito), rabbit anti-SMYD3 (Abcam, ab187149), rabbit anti-SMYD3 (Diagenode, C15410253-100), rabbit anti-Cyclin D1 (Abcam, ab 16663), rabbit anti-P21 (ab 109199), mouse anti-MYOGENIN (F5D ab 1835).

The primary antibodies below were utilized: myosin heavy chain (MHC; R&D Systems, USA, MAB4470), slow MHC (Abcam, ab185967), fast MHC (Abcam, ab91506), MHC IIa (Abcam, ab124937), MHC IIb (Abcam, ab221149), myoglobin (Santa Cruz, USA, sc-393020), troponin I-ss (Santa Cruz, sc-514899), TOM20 (Abcam, UK, ab186735), TIM23 (Abcam, ab230253), SESN2 (Abcam, ab178518), AMPK (Cell Signaling Technology [CST], USA, 5831), phospho-AMPK (CST, 50081), PGC-1α (Abcam, ab106814), HIF-2α (Abcam, ab109616), GAPDH (Protein Technology, PRC, 60004-1-Ig). The primary antibody dilution factors were 1:3000 (R&D Systems), 1:5000 (Protein Technology), or 1:1000 (Abcam and CST).
In short, the protein was abstracted via RIPA lysis buffering solution to which 1% protease suppressor was added (Roche, USA, 11206893001). The same quantity of protein (10–50 μg) was isolated via 10% SDS–PAGE, and afterwards moved onto nitrocellulose films (Merck Millipore, USA, Z358657). The films were subjected to blockade with 5% w/v BSA prior to cultivation with the first antibodies under 4 °C overnight. Subsequently, the films were cultivated with second antibodies conjugated with antirabbit or antimouse IgG (Abcam) for 60 min under ambient temperature and visualized via the Immobilon ECL matrix tool (Merck Millipore, WBKLS0050).