Sc 28534

To isolate the DGC and associated proteins, 100 μg of whole cardiomyocytes extract was loaded on a 10-40% glycerol gradient and centrifuged for 24 h at 35,000 r.p.m. using a MLS-50 Swinging-Bucket rotor (Beckman Coulter). Fractions collected from glycerol gradients were subjected to trichloroacetic acid (TCA) precipitation (10%) and precipitates were analysed by SDS–PAGE and immunoblotting. The following primary antibodies were used: anti-rat recombinant Agrn (MAB550, R&D systems), anti-α-dystroglycan (sc-28534, Santa Cruz), anti-β-dystroglycan (Mandag2[7d11], DSHB), anti-syntrophin (sc-50460, Santa Cruz), anti-dystrophin (ab15277, Abcam), anti-Grb2 (sc-8034, Santa Cruz), anti-Myh6 (ab50967, Abcam), anti-actin (A2066, Sigma-Aldrich), anti-Actn1 (A2543, Sigma-Aldrich), anti-Des (ab8592, Abcam), anti-Yap (sc-15407, Santa Cruz) and anti-H3f3a (ab62642, Abcam). Horseradish peroxidase anti-mouse, anti-rabbit or anti-goat (Sigma-Aldrich) was used as secondary antibody.

Adherent cells were grown on a gelatin-coated 96-well plate. The cells were fixed with 4% PFA in PBS for 10 min and permeabilized with 0.2% Triton X-100 in PBS for 5 min, and blocked with PBS containing 0.1% Triton and 3% bovine serum albumin (BSA) for 1 h at room temperature. For immunostaining, the cells were incubated for 2 h with the following monoclonal antibodies diluted in the blocking solution: anti-cTnT (1:200, ab33589, Abcam), anti-cTnI (1:200, ab47003, Abcam), anti-Nkx2.5 (1:300, sc-8697, Santa Cruz), anti-Dag1 (1:200, sc-28534, Santa Cruz) antibodies were used to identify cardiomyocytes; anti-Ki67 antibody (1:200, 275R, Cell Marque), anti-phosphorylated histone 3 (pH3) (1:200, SC-8656-R, Santa Cruz Biotechnology) and anti-aurora kinase B (AURKB, 1:100, 611082, BD Transduction Laboratories) antibodies were used to analyse cell-cycle re-entry, DNA synthesis, karyokinesis and cytokinesis, respectively. Cells were then washed three times with PBS and stained for 45 min at room temperature with suitable secondary antibody. This was followed by 5 min of DAPI (4,6-diamidino-2-phenylindole dihydrochloride). The cells were viewed under Nikon fluorescence or an Olympus live-cell imaging microscope.