Revert 700 total protein

Sera protein concentration was measured with Pierce^TM BCA Protein Assay (Thermo Fisher Scientific, Les Ulis, France). Protein extracts were separated on 4–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific), and transferred to a nitrocellulose membrane using the iBlot2 Dry Blotting system (Thermo Fisher Scientific) for 8 min 30 at 20 V. Total protein on blotted membranes were determined with the Revert 700 Total Protein (LI-COR Biosciences, Lincoln, NE) staining. Membranes were washed, blocked in Odyssey Blocking Buffer (LI-COR), 1 h, room temperature. Primary antibody (MYOM3 17692−1-AP, Proteintech 1/500 in 50% blocking buffer) was incubated overnight at 4 °C. After washings in 1× TBST (Tris/HCl (pH 7.5—20 mM)/NaCl (150 mM)/Tween 20 (0.1%)), the diluted secondary antibodies (1/1000 in 50% Odyssey Blocking Buffer) were incubated 1 h at room temperature. Membrane images were acquired with the Odyssey Infrared Imaging System. Band density was quantified using Image Studio Lite 4.0 software (LI-COR Biosciences, Lincoln, NE, USA) and normalized to total protein values.

Proteins were extracted from tissues or cells by RIPA buffer (Thermo Fisher Scientific) supplemented with Protease Inhibitor Cocktails (Complete PIC; Roche) and Benzonase 1:1,000 (Millipore). Total protein concentration was measured by using Pierce BCA Protein Assay (Thermo Fisher Scientific). Equal amounts of protein were then loaded and separated by precast 4–12% Bis-Tris polyacrylamide gel (Thermo Fisher Scientific). Subsequently, the protein was transferred to a nitrocellulose membrane with the iBlot2 Dry Blotting system (Thermo Fisher Scientific). For detecting the proteins of interest, the membrane was blocked in Odyssey Blocking Buffer (LI-COR) for 1 h at room temperature before incubated with primary antibodies diluted in 50% Odyssey Blocking Buffer overnight at 4°C. After washed in 1× PBST, the membrane was incubated with secondary antibodies 1/5,000 diluted in the 50% Odyssey Blocking Buffer for 1 h at room temperature. The membrane was then rewashed and blotting signals were acquired in the Odyssey Infrared Imaging system. Total protein (for muscle samples) measured by Revert 700 Total Protein (LI-COR) (for muscle samples) or Actin (for in vitro cells) was used as loading controls for quantification. Details of antibodies used in this study are presented in Table S4.