“…Protein concentration was determined by the BCA Protein Assay Kit (Thermo Fisher Scientific), and 30â40 Îźg of each protein sample was subjected to SDS-PAGE in polyacrylamide Novex NuPAGE Bis-Tris 4â12% gels (Thermo Fisher Scientific), followed by electrotransfer onto polyvinylidene difluoride membranes (Millipore), as previously described ( Metti et al, 2020 ). Membranes were stained with Ponceau (Sigma-Aldrich) and blocked for 1 h with 5% non-fat dry milk (Bio-Rad, Hercules, CA, USA) in Tris-buffered saline solution containing 0.1% Tween 20 (TBS-T), and then incubated overnight at 4°C with the primary antibodies, as previously described ( Gambarotto et al, 2022 ). The following primary antibodies were used: mouse monoclonal anti-MyH3 (1:500; F1.652; Developmental Studies Hybridoma Bank, Iowa City, IA, USA), mouse monoclonal anti-MyoG (1:300; sc-12732; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit polyclonal anti-phospho-GSK-3Îą/β (Ser 21/9) (1:1000; 9331S; Cell Signaling Technology, Danvers, MA, USA), rabbit monoclonal anti-β-catenin (1:500; ab32572; Abcam, Cambridge, UK), and mouse monoclonal anti-β-actin (1:3000; A5316; Sigma-Aldrich).…”