Cx40 a

Immunofluorescence assay was performed as previously described^{34 (link)} for high content screening analysis (IN Cell Analyzer 2200, GE) and confocal microscopy (Multiphoton Microscope LSM 780 NLO, Zeiss). Cells were fixed with 4% paraformaldehyde (PFA) for 60 min at room temperature, washed twice with PBS, permeabilized with 0.1% Triton for 15 min, and blocked with 5% Bovine Serum Albumin (BSA). Primary antibodies against the following proteins were used: 1:400 cardiac troponin I (4T21/2, HyTest), 1:200 cardiac troponin T (MA512960, Thermo Fisher), 1:900 vimentin (AB92547, Abcam), 1:750 connexin 43 (AB11370, Abcam), 1:100 connexin 40 (CX-40A, Alpha diagnostic International), 1:100 contactin-2 (AF4439, R&D Systems), 1:100 NAV1.5/Scn5a (sc-271255, Santa Cruz), and 1:200 ki67 (AB16667, Abcam). All primary antibodies were diluted in 2% BSA and incubated overnight at 4 °C. For the high content screening analysis, ten fields of each sample were collected and analyzed by the software IN Carta (GE Healthcare Life Sciences). The high throughput analysis retrieved phenotypic information of more than 500 single-cells per sample.

Ventricular tissue was collected at 7 weeks of age and homogenized in lysis buffer (150 mM sodium chloride, 1.0% Triton X‐100, 0.5% sodium deoxycholate, 0.1% Sodium dodecyl sulfate, 50 mM Tris, pH 8.0) using silica beads and a homogenizer (BioSpec). Lysates were measured using the Pierce BCA assay kit (Thermo Scientific #23225) and normalized to 10 µg total protein. Lysates were separated using polyacrylamide gel electrophoresis and gel was transferred to nitrocellulose (NC) membranes. Membranes were blocked in 5% BSA prepared in 1x TBS‐T. After blocking, NC membranes were incubated with primary antibodies Cx40 (1:1000, Cx40‐A, Alpha Diagnostic Intl Inc.), Connexin 43 (1:1000, Cx43, Sigma C6219), or Contactin 2 (1:1000, R&D Systems, AF4439). Membranes were incubated with secondary antibody goat anti‐rabbit IgG‐HRP (1:1000, Santa Cruz Biotechnology sc‐2004) or donkey anti‐goat IgG‐HRP (1:1000, Santa Cruz Biotechnology sc‐2020). Signals were detected using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Scientific #34577). Total protein was determined using Memcode Reversible Protein Stain Kit (Thermo Scientific #24580).