Mouse anti actinin

For pluripotency marker stainings, iPSC colonies were passaged as described above and grown on Matrigel^TM-coated coverslips in ES medium containing, 50% MEF-conditioned media (own preparation) supplemented with 5 ng/ml FGF2 (Sigma). Colonies were then stained for alkaline phosphatase according to the manufacturer’s protocol (Millipore) or were fixed with 4% paraformaldehyde (PFA) in PBS for 10 min at RT for analysis of pluripotency markers by immunofluorescence. Fixed colonies were incubated for 2 h in blocking solution (3% normal horse serum and 0.05–0.2% Triton-X100 in PBS). Plates were incubated over night at 4 °C using the following primary antibodies: rabbit anti-Nanog (1:500), rabbit anti-Oct4 (1:1000), mouse anti-SSEA4 (1:500), mouse anti TRA-1-60 (1:500) (all from Abcam) and mouse anti-Sox2 (1:500, R&D Systems). Differentiated EBs were stained with rabbit anti-α-SMA (1:500, Sigma Aldrich), mouse anti-α-Fetoprotein (1:500, Abcam), rabbit anti-GATA4 (1:500, Abcam), and mouse anti-TUJ1 (1:1000, Covance), mouse anti-Actinin (Sigma, 1:200) and mouse anti Beta-Catenin (BD Bioscience 1:500).

Total protein was extracted from the patient’s cardiac and skeletal muscle specimens using RIPA buffer containing protease inhibitors (Roche, Indianapolis, IN, USA). The lysates were sonicated on ice and centrifuged at 20,000 × rpm for 30 min at 4 °C. The supernatant was collected, and protein concentrations were determined employing a BCA protein assay kit (Thermo Fisher Scientific). After mixing with NuPAGE LDS Sample Buffer (Thermo Fisher Scientific), cell lysates were denatured at 70 °C for 10 min, electrophoresed utilizing a NuPAGE Novex Tris-acetate gel 3–8% (Invitrogen) at 150 V for 70 min, and then transferred to PVDF membranes. The membranes were incubated with primary antibodies, followed by incubation with a secondary antibody using the iBind Flex Western Device (Thermo Fisher Scientific). The following primary antibodies were utilized: rabbit antidystrophin (1:500, Abcam, UK; ab15277), mouse antiactinin (1:1000, Sigma-Aldrich, UK; A7811) and antitubulin antibody (1:1000, Sigma-Aldrich, UK; T6199); Histofine Simple Stain MAX-PO (1:100, NICHIREI BIOSCIENCE INC., Tokyo, Japan; 424151) was used as a secondary antibody. Proteins were detected utilizing the ECL Prime Western Blotting Detection Reagent (GE Healthcare, UK; RPN2232), and a ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA). The data were analyzed with Image Lab 6.0 (Bio-Rad).