Anti igf1

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).

Tissues were homogenized as described (Brüning et al., 1998 (link)). Western blot analyses of insulin signalling proteins were performed on liver homogenates as previously described (Valverde et al., 1998 (link)). For immunoprecipitation, after protein content determination, equal amounts of protein (400 μg) were immunoprecipitated overnight at 4°C with anti-GLUT-2 antibodies (C-19) (1:500; sc-7580, Santa Cruz Biotechnology, Dallas, TX). The immune complexes were collected on protein A-agarose beads and submitted to SDS-PAGE. For western blot, the antibodies used were anti-insulin receptor β subunit (1:1000; sc-711), anti-GLUT-2 (H-67) (1:500; sc-9117) and anti-IGF-I (1:200; sc-9013) (all from Santa Cruz Biotechnology) and anti-β-actin (1:5000; A2228, Sigma-Aldrich, St. Louis, MO). Rabbit and mouse primary antibodies were immunodetected using horseradish peroxidase-conjugated polyclonal anti-rabbit or mouse antibodies, respectively (1:5000; NA931V and NA934V, GE Healthcare, Buckinghamshire, UK). Loading was normalized by β-actin. The band intensities were quantified using ImageJ software (http://rsb.info.nih.gov/ij).