Protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3

The peripheral parts of muscle specimens were immediately frozen in liquid nitrogen and stored at −80°C for biochemical studies. Western blotting was performed as previously described (Fujimura and Usuki, 2015 (link), 2017 (link)). Briefly, the samples were sonicated for 5 s in tissue lysis buffer (T-PER Mammalian Protein Extraction Reagent; Pierce Biotechnology, Rockford, USA) containing Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma-Aldrich, St Louis, USA). The samples were centrifuged (14,000 g for 1 h), and the supernatants were collected. The protein content was determined using the DC Protein Assay Kit II (Bio-Rad Laboratories, Hercules, USA). The cell lysates (20 μg protein) were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 10% gel (Tefco, Tokyo, Japan) and transferred to nitrocellulose membranes (GE Healthcare, Buckinghamshire, UK). The membranes were then subjected to the following antibody probes: anti-MGF (Millipore, Billerica, USA); anti-IGF-I (Santa Cruz Biotechnology, CA, USA); anti-YAP1 (Novus Biologicals, Centennial, CO, USA); anti-phospho-YAP1 (Abcam, Cambridge, UK); anti-PAX7 (Cytoskeleton Incorporated, Denver, USA); anti-phospho-ERK, anti-ERK, anti-AKT, anti-phospho-AKT, anti-phospho-4EBP1, and anti-phospho-p70 S6 kinase (Cell Signaling Technologies); and anti-β-actin (Sigma-Aldrich).

Cell lysates were prepared using RIPA buffer (Sigma Aldrich, St. Louis, MO) with Protease Inhibitor Cocktail and Phosphatase Inhibitor Cocktail 2 and 3 (Sigma Aldrich, St. Louis, MO) and centrifugation at 12,000 X g for 15 min at 4°C. Protein concentration was determined using the Bradford protein assay reagent (Bio-Rad, Hercules, CA). Lysates were separated by 4-20% gradient sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Protein was transferred onto a polyvinyl difluoride (PVDF) membrane. Membranes were incubated overnight at 4°C with primary antibody followed by incubation with horseradish peroxidase secondary antibody. Enhanced chemiluminescence (ECL) reagent was added to the membrane according to the manufacturer's protocol (Pierce Biotechnology Inc., Rockford, IL). Chemiluminescence was detected on the ChemiDoc™ Imaging System, and densitometry was performed using the Image Lab software (Bio-rad, Hercules, CA)

The total protein content was extracted from RAW 264.7 cells using RIPA buffer (Biosolution, Seoul, Korea) with protease inhibitor cocktail and phosphatase inhibitor cocktail 2 and 3 (Sigma-Aldrich). The collected protein samples were quantified using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Equivalent amounts of proteins (20 μg) were separated via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane. The membrane was incubated with primary antibodies after blocking with 5% skim milk (Difco Laboratories) for 1 h; subsequent washing and incubation with horseradish peroxidase-conjugated secondary antibodies followed. The separated proteins were detected using ECL Plus Western blotting detection reagents (Amersham Bioscience, Buckinghamshire, UK) using ChemiDoc™ Imaging Systems (Bio-Rad). The acquired images were analyzed and quantified using the Image Lab™ Software (Bio-Rad).