Anti tbx5

EB dissociation was performed as described above for flow cytometry. Cells were replated onto gelatin-coated four-well chamber slides and allowed to attach for 18-24 h. Slides were fixed in 4% paraformaldehyde for 15 min, gently washed three times for 5 min in phosphate buffered saline (PBS) and blocked with either cytosolic antibody staining buffer (CASB) (PBS, 1% FBS, 0.1% BSA, and 0.1% TritonX-100) or nuclear antibody staining buffer (NASB) (1% FBS, 0.2% BSA, and 0.25% Triton-X-100) for 1 h. Cells were incubated with primary antibodies diluted in blocking buffer overnight. Cells were washed with PBS three times for 5 min and then incubated with Alexa Fluor-labeled secondary antibodies for 1 h at room temperature. Three additional PBS washes were performed with DAPI added to the second wash. Coverslips were added and sealed with acrylic. Unless otherwise stated in the text, images were obtained using a Zeiss AxioImager microscopy. The primary antibodies were used as follow: anti-Cx43 (Sigma-Aldrich, #C6219, 1:1000), anti-CT3 (DSHB, #CT3, 1:250), anti-DsRed (Clontech, #632392, 1:500), anti-GFP (ThermoFisher Scientific, #A11122, 1:500), anti-HCN4 (Sigma-Aldrich, #SAB520035, 1:1000), anti-Shox2 (Abcam, #ab55740, 1:1000), anti-Tbx5 (ThermoFisher Scientific, #42-6500, 1:500), asnti- Islet1 (Abcam, #ab20670, 1:500).

Protein was extracted from different cell lines in RIPA buffer with protease inhibitors and sodium orthovanadate. Total protein was quantified by Bradford assay (Pierce Coomassie Protein Assay Kit) compared to a serial dilution of BSA in a range of 0.064-2 mg/ml. For western blots, 20 μg/protein was loaded per well into prepared SDS-PAGE gels (Invitrogen NuPAGE 12% Bis-Tris gels). Proteins were transferred to PVDF membranes and immunoblotted for visualization with Immobilon western chemiluminescent Horse radish peroxidase (HRP) substrate (Millipore) using a GeneGnome XRQ-chemiluminescence imaging system. Quantification was based on raw peak volume data normalized to the raw peak value for GAPDH. Antibodies used were anti-Tbx5 (ThermoFisher Scientific, #42-6500, 1:500), anit-TAK1 (Sigma-Aldrich, #AB1305414, 1:1000), anti-HCN4 (Invitrogen, MA3-903, 1:1000), HRP conjugated anti-GAPDH (Invitrogen, #MA5-31457, 1:5000) and anti-Cx43 (Sigma-Aldrich, #C6219, 1:1000). Statistical significance was determined by student t-test with P<0.05 considered significant.