Quantitative Western Blot Analysis

For Western blot analysis, half tibialis anterior muscles were snap-frozen, lysed using lysis buffer [50 mM Tris-HCl pH 7.8, 150 mM NaCl, 1% NP-40 (AppliChem/A1694), 10% glycerol, 5 mM EDTA, 1 mM EGTA, 1 Halt Protease and Phosphatase Single-Use Inhibitor Cocktail (FisherScientific/10025743), and 0.5 mM PMSF (AppliChem/A0999), pH adjusted to 7.4], and subjected to SDS-PAGE followed by Western blot analysis as already described (Wild et al., 2016 (link); Straka et al., 2018 (link)). In each lane, equal amounts of protein were loaded (20 μg). Chemiluminescence signals were obtained using an ECL system (Biozym Scientific GmbH/541004) in combination with a Syngene G:Box Chemi XX6 chemiluminescence imager (Thermo Fisher Scientific, Schwerte, Germany). The analysis used ImageJ freeware image processing software².