Anti mef2c

Total protein was prepared in ice-cold RIPA buffer (150 mM Tris-HCl, pH = 7.6, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na₃VO₄) containing a cocktail of protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) and then quantified using a BCA Protein assay kit (Thermo Fisher Scientific). Protein extracts were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The following primary antibodies were used: anti-ANP (Abcam, Cambridge, UK; dilution 1:1,000), anti-BNP (Abcam; dilution 1:500), anti-β-MHC (Abcam; dilution 1:1,000), anti-MEF2C (Abcam; dilution 1:1,000) and anti-β-actin (Abcam; dilution 1:3,000). The horseradish peroxidase-conjugated anti-rabbit (Abcam; dilution 1:5,000) or anti-mouse (Abcam; dilution 1:5,000) IgG was used as a secondary antibody. Protein bands were detected using the enhanced chemiluminescence (ECL) detection kit (Immobilon Western Chemiluminescent HRP Substrate, Millipore) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Nucleoproteins were extracted using a nuclear extraction kit (Merck Millipore), separated, subjected to SDS‐PAGE and then transferred to polyvinylidene difluoride membranes using a Bio‐Rad semidry electrotransfer apparatus. The nitrocellulose membranes were blocked with 5% non‐fat milk in Tris‐buffered saline and incubated with monoclonal antibodies (anti‐G9α, anti‐H3K9me3, anti‐MEF2C, anti‐Cx43, anti‐ANP, anti‐β‐MHC and anti‐GAPDH [Abcam]) diluted in Tris‐buffered saline. Protein bands on immunoblots were visualized by enhanced chemiluminescence. After scanning, bands were quantified using Quantity One software version 4.4 (Bio‐Rad).

Immunofluorescence staining was performed on cryosections as previously described (Han et al., 2014 (link)). The primary antibodies used in this study were: anti-EGFP (Abcam), anti-pERK (Cell Signaling Technology), anti-pTie2 (Tyr992) (Millipore), anti-Mef2c (Abcam), anti-MF20 (DSHB), anti-BrdU (Sigma), anti-α-actinin (Sigma), anti-CD31(Abcam), anti-Angpt4 (Invitrogen), anti cTnT (Abcam), anti-Ki67 (Abcam), and anti-Aurora B (Abcam). The secondary antibodies used in this study were: Alexa Fluor 488 goat anti-mouse IgG (Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen), Alexa Fluor 647 goat anti-chicken IgY H&L (Abcam). Nikon A1 confocal microscope and Zeiss Axio Scan were used to observe and record the immunostaining images. To quantify the pERK and pTie2 signals, heart area and pERK/pTie2 positive area were analyzed using Surface function in Imaris, then we calculated the ratio of pERK/pTie2 positive area/heart section area.