Mlc2a

After the cells were allowed to differentiate for 15 days, they were washed with PBS, fixed in 4 % paraformaldehyde for 15 min, permeabilized with 0.5 % Triton-X 100 for 10 min and blocked with 5 % bovine serum albumin (BSA; Sigma-Aldrich, St. Louis, MO, USA) at 37 °C for 30 min. Subsequently, the cells were first incubated with mouse monoclonal anti-cardiac troponin T antibody (cTnT, 1:100; Abcam, Cambridge, MA, USA) for 90 min, followed by staining with goat anti-mouse IgG-FITC (1:400; Santa Cruz Biotechnology, Dallas, TX, USA) for 60 min at room temperature. After the cells were washed three times with PBS to remove the excess staining, they were incubated with rabbit polyclonal anti-myosin light chain (MLC2a, 1:100, Santa Cruz Biotechnology Inc) primary antibody for 90 min at room temperature. This incubation was followed by incubation with goat anti-rabbit IgG-TR (1:400; Santa Cruz) for 60 min at room temperature. After the cells were rinsed with PBS, the nuclei were counterstained with 4’,6-diamidino-2-phenylindole (DAPI; 1:1000; Sigma, St. Louis, MO, USA) for 5 min. The samples were finally imaged using a fluorescence microscope (IX71, Olympus, Tokyo, Japan).

Total protein was extracted from cells underwent differentiation for 15 days. The primary antibodies as follows: α-MHC,β-MHC (1:1,000; Abcam, Cambridge, MA, United States), MLC2a, MLC2v (1:1,000, Santa Cruz Biotechnology, Dallas, TX, United States) and GAPDH (1:10,000; Abcam, Cambridge, MA, United States). The band intensities were quantified using ImageJ software.

Mouse embryos were generated and obtained in collaboration with P.Uhrin (Vienna). After staging of pregnancy the dams were killed and embryos harvested and fixed. Serial sections were immunostained for the myocardial marker MLC2-a (1/6000, kindly provided by S.W.Kubalak, Charleston, SC, USA) and WT-1 (1/1000, Santa Cruz Biotechnology, CA, USA) as previously described [7] (link).