Sc 7392

Frozen TA muscles were cut transversally, fixed in 4% PFA for 20 min and permeabilized with methanol for 6 min at −20 °C. Muscle sections were then blocked with a solution containing 4% BSA in PBS followed by incubation with anti-mouse AffiniPure Fab fragment (Jackson) to avoid unspecific binding. Immunostaining with primary antibodies was performed overnight at 4 °C. Secondary antibodies coupled to Alexa Fluor 488 or 594 (Molecular Probes) were used to reveal antibody binding. Nuclei were visualized by counter staining with DAPI. Cultured cells were fixed in 4% PFA, permeabilized with either 0.25% Triton (for phopho-p38 antibody) or methanol and blocked with 4% BSA in PBS before antibody incubation. Immunostaining and detection was performed as above. Primary antibodies used were: anti-laminin (Sigma, L9393; dilution 1:1,000), anti-PJA1 (Proteintech, 17687-1-AP; dilution 1:50), anti-MYOD (BD, 554130; dilution 1:50), anti-EZH2 (AC22, Cell Signaling; dilution 1:150), anti-MyHC (MF-20, DSHB; dilution 1:30 and Santa Cruz, SC-20641; dilution 1:100), anti-KI67 (BD 556003; dilution 1:1.000), anti-HA (Santa Cruz, SC-805 and SC-7392; dilution 1:50) and anti-phospho-p38 (Cell Signalling, D3F9; dilution 1:150). Images were acquired with a Leica confocal microscope and edited using the Photoshop CS4 software. Fields reported are representative of all examined fields.

The proximity ligation assay was conducted using a PLA kit according to the manufacturer’s protocol. After fixation with PFA, cells were incubated with mouse anti-HA (Santa Cruz, sc-7392) and rabbit anti-GSK3B (Cell Signaling, 9315S) antibodies. Following primary antibody incubation, a pair of PLA probes (Sigma, DUO92002 and DUO92004) were added, and probe ligation, signal amplification (Sigma, DUO92007), and mounting (Sigma, DUO82040) were performed according to the manufacturer’s instructions. Representative images of the PLA signal were obtained using a confocal microscope, and the number of PLA puncta per cell was counted.