Biorruptor pico

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.

Sciatic nerves were homogenized at 4°C in RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate SDS, and 5 mM EGTA) containing protease inhibitors (Mini Protease Inhibitor Cocktail; Sigma-Aldrich) and phosphatase inhibitors (Phosphatase Inhibitor Mini Tablets; Fisher Scientific). We homogenized the tissue using Bullet Blender Homogenizer BBX24-CE (Next Advance) and then sonicated for 4 min (30 s on/off) using a Biorruptor Pico (Diagenode). Protein concentrations were determined by the BCA method (Thermo Scientific). 10 μg of total protein was subjected to SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and blotted on to Protran nitrocellulose membrane (Amersham Biosciences). Membranes were blocked using 5% milk (Sigma-Aldrich) in TBS 1% and incubated for 16 hr at 4°C with the indicated primary antibody, washed and incubated with secondary antibodies, and developed with ECL Prime (Amersham). Antibodies used can be found online (Key Resources Table). We used an Amersham Imager 680 machine (Amersham) for visualization. Measurements from the proteins of interest were normalized to loading control GAPDH and/or CALNEXIN. When normalized to both loading controls, a mean between the normalization with GAPDH and the normalization with CALNEXIN was used for analysis. The whole membrane Western blot images are shown in source data file four online.