Mlc 2v

The cell lysates were prepared in lysis buffer (100 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.1% SDS and 1% Triton X-100) at 4°C. The extracts were separated by SDS-PAGE, transferred onto a polyvinylidine difluoride membrane (Millipore, MA, USA) and detected using antibodies against PARP, LC3 (Cell Signaling Technology, MA, USA), α-actinin (ACTN), p53, p21, MLC-2v, H3P, HuR, Twist, CACNb1 (Santa Cruz Biotechnology, CA, USA), γH2A.X, myogenin (Epitomics, CA, USA) and hemagglutinin (HA) (3F10, Hoffmann-La Roche, Basel, Switzerland).

Total protein was extracted from cells and protein concentration was analyzed by a NanoDrop 2000 spectrophotometer (Thermo Scientific, Delaware, USA). For western blot analysis, 50 µg of proteins were separated and transferred onto polyvinylidene difluoride membrane (PVDF; Millipore, Billerica, MA, USA). Following blocking for 1 h in PBS with 0.1% Tween 20 (PBST) and 5% BSA, the membranes containing proteins of interest were incubated overnight with specified primary antibody at 4 °C. PVDF membranes were washed in TBST and incubated with secondary antibodies labeled with HRP and detected by ECL (Pierce, Rockford, IL, USA). Antibodies used in this study are Nkx-2.5 (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), GATA4 (1:500; Santa Cruz Biotechnology), α-MHC (1:500; Santa Cruz Biotechnology), MLC-2v (1:500; Santa Cruz Biotechnology), Sox6 (1:10,000; CST, Inc., Danvers, MA, USA), and β-actin (1:10,000; CST, Inc.).

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.