C 21x sc313x

Protein extracts of neonatal rat cardiomyocytes were prepared with RIPA buffer. Protein extracts (1 mg) were immunoprecipitated overnight at 4°C with 5 μg of anti-NFAT (ab 2722, Abcam, Cambridge, UK) or 5 μg of anti-MEF-2c (C-21X sc313x, Santa Cruz Biotechnology Inc., CA, USA) antibodies. Immunoprecipitated proteins were recovered using Protein A Sepharose CL-4B (17-0780-01, GE Health Care, IL, USA). Immunoprecipitated proteins were analyzed by Western blot with anti-MEF-2c (C-21X sc313x, Santa Cruz Biotechnology Inc., CA, USA) antibody.

Primary cultures of neonatal rat cardiomyocytes were used for ChIP experiments. The cross-linking reaction was done using 1% formaldehyde for 15 min. The reaction was stopped with glycine (Sigma-Aldrich, MO, USA) at a final concentration of 0.125 M. Culture medium was removed, and the cells were washed with PBS 1X with PMSF 1 mM. The cells were then lysed with lysis buffer (Tris-HCl 50 mM pH 8.0, EDTA 10 mM, SDS 1%, and Sigma Fast Protease Inhibitor Cocktail [Sigma-Aldrich, MO, USA]). The cells were subjected to 5 sonication cycles of 15 sec ON with 90 sec OFF in a Biorruptor Pico (Diagenode, NJ, USA) sonication device. Immunoprecipitation was done with One-Day ChIP Kit (Catalog number C01010081, Diagenode, NJ, USA) following the manufacturer instructions. Sonicated chromatin was incubated with 8 μg of antibody against MEF-2c (C-21X sc313x, Santa Cruz Biotechnology Inc., CA, USA), NFATc3 (M75X sc8321, Santa Cruz Biotechnology Inc., CA, USA) or Sp1 (PEP-2 sc59x, Santa Cruz Biotechnology Inc., CA, USA) as a negative control. The primers used for PCR reaction are listed in S1 Table. The PCR reaction cycles were as follows: 10 min at 95°C, 40 cycles (30 sec at 95°C, 30 sec at 60°C, 30 sec at 72°C) and 5 min at 72°C. The final reaction volume was 20 μL.