Anti mef2

Primary antibodies used were anti-PCNA (1:2 000; Dako #M0879), anti-mef2 (1:50; Santa Cruz Biotechnology #sc-313) and anti-H3P (1:1 000, Cell Signaling Technology #9706). DAPI was used to stain nuclei. Images shown are single optical planes acquired using a Leica SP5 confocal microscope. For quantification of PCNA positive cardiomyocytes, the percentage of Mef2-positive cells also positive for PCNA was quantified within a zone that extended 150 μm from the wound border. For each heart 2-3 sections displaying the biggest wounds were analyzed. H3P-positive cardiomyocytes were quantified on all heart sections, which contained an injury area (average eight sections per heart). In this case the total cardiomyocyte number in the border zone was estimated by determining the average density of cardiomyocytes per μm² in three separate areas (size: 75 μm²) and by multiplying this number by the total border area size.

HEK293T cells seeded in 10cm or 6cm plates. Cells were transfected with 10 μg pcDNA3-myc (empty vector) or pcDNA3-myc-EGR1 and 5 μg of pCMV-MEF2A-Flag. 36–48 hours post-transfection, protein was harvested in AT buffer (20% glycerol, 1% Triton X-100, 20 mM HEPES pH7.9, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, 1 μg/mL PMSF, and 1:25 protease inhibitor mixture (Roche). Approximately 35 μL Protein G sepharose beads (GE Healthcare) and 1 μg of anti-Myc was added and incubated with AT buffer precursor (AT buffer excluding DTT, PMSF, and protease inhibitors). The beads, protein, and antibodies were incubated at 4°C, rotating overnight. Samples were boiled samples and loaded onto an 8% SDS-PAGE Gel. A western blot was then run as outlined below, using 1:2,000 anti-Flag as a primary antibody.
Western blots were performed as previously described [15 (link)]. Primary antibodies used were as follows: 1:10,000 anti-Flag (Sigma), 1:2,000 anti-c-myc (Santa Cruz Biotechnology), 1:2,000 anti-MEF2 (Santa Cruz Biotechnology), and 1:2,000 anti-GAPDH (Santa Cruz Biotechnology). Blots were incubated with horseradish peroxidase-conjugated secondary antibodies (1:10,000) and reacted with western lighting chemiluminescent reagent (Perkin Elmer) and subsequently exposed to Blue Lite AutoRad film (BioExpress).

Whole-mount hearts were extracted, fixed and cryosectioned. All adult heart cryosections had 10 µm thickness. The following primary antibodies were used in this study: anti-Mef2 (Santa Cruz, Dallas, TX, USA); anti-Abcc6 (Santa Cruz); anti-cTnT (DSHB, Iowa City, IA, USA); anti-MF20 (MHC; DSHB); anti-Collagen Ia (Abcam, Cambridge, CB2 0AX, UK); anti-Fibronectin (Sigma-Aldrich, Saint Louis, MO, USA). Secondary antibodies included Alexa Fluor 488 goat against mouse IgG (H + L) (1:1000; Life Technologies, A11001;Thermo Fisher Scientific, Carlsbad, CA, USA), Alexa Fluor 594 goat against mouse IgG (H + L) (1:1000; Life Technologies, A11005), Alexa Fluor 594 goat against rabbit IgG (H + L) (1:1000; Life Technologies, A11012) and Alexa Fluor 488 goat anti-rabbit (1:1,000; Life Technologies, A11008). Confocal images were obtained using a Zeiss LSM710 laser scanning confocal microscope.
To quantify the cardiomyocyte number, one section showing the largest area was selected from each heart, and images were taken using a 10× objective. The number of Mef2+ was manually counted using Image J software within the same region of the ventricle. WT and mutant hearts were selected for statistical count. Nine cardiac cryosections were taken from WT and mutant hearts for statistical count.