Anti cx43

Western blot analysis was performed as previously described [50 (link)] using 50 µg of protein from each sample for separation by sodium dodecyl sulphate-gel electrophoresis and following antibodies: rabbit polyclonal anti-Phopsho-JNK (Cell Signaling, Cambridge, UK), rabbit polyclonal anti-JNK (Cell Signaling), rabbit polyclonal anti-Cx43 (Sigma), and mouse anti-ß-Actin (Sigma). Densitometric quantification and normalization to the corresponding beta-actin levels was performed using Fiji software.

Western blotting was performed as described before (Höpfner et al., 2004). The following antibodies were used: anti‐Cx43 (Sigma), anti‐tubulin (Thermo Fisher), anti‐GAPDH (Calbiochem; Merck), anti‐bisphosphoglycerate mutase (BPGM; Novus Biologicals, Littleton CO, USA), anti‐tubulin ß‐2B, (OriGene, Rockville MD, USA), anti‐GLUT1 (Thermo Fisher), and secondary horseradish‐peroxidase‐coupled antibodies (Vector Laboratories, Burlingame, CA, USA). Membranes were developed using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare Life Sciences, Freiburg, Germany) and a Fusion SL camera (Vilber Lourmat, Eberhardzell, Germany). For quantification, imagej was used and the density of the protein bands was either normalized to tubulin (GLUT‐1, BPGM) or GAPDH (Cx43) loading control.

Immunofluorescence and immunohistochemistry assays were performed as previously described [2 (link), 10 (link)]. The following primary antibodies were used: anti-Cx43 (Sigma-Aldrich, C6129), anti-collagen II (Invitrogen, Thermo Fisher Scientific, MA5-12789), anti-Ki-67 (BD, 550609), anti-Twist-1 (sc-81417) and anti-NF-κB (sc-8008) from Santa Cruz Biotechnology. Goat anti-rabbit FITC-conjugated (F-2765, Invitrogen, Thermo Fisher Scientific) and goat anti-mouse Alexa 594-conjugated (A-11032, Invitrogen, Thermo Fisher Scientific) secondary antibodies were used.

Y27632, Lucifer yellow and anti-Cx43 antibodies were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-RhoA, anti-Rac1, anti-Cdc42, anti-RhoGDI, and anti-cofilin antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The anti-p190RhoGAP antibody was purchased from Millipore (Lake Placid, NY, USA). Anti-phospho-RhoA (Ser188), anti-phospho-LIMK1/2 (Thr508 and Thr505), anti-LIMK1, and anti-Cx43 antibodies were purchased from Abcam (Cambridge, MA, USA). The anti-phospho-cofilin antibody (Ser3) was obtained from Cell Signaling Technology (Danvers, MA, USA).

Hearts were rapidly excised, perfused and equilibrated and homogenized as above. S-nitrosylated proteins were detected by the biotin-switch method as previously described [31 (link)]. First, free thiols (-SH) in tissue homogenates were blocked for 1 h at 50°C in the dark with methyl methanethiosulfonate (MMTS). Proteins were precipitated with 4 volumes of ice-cold acetone, washed repeatedly with acetone to remove free MMTS and resolubilized. Thereafter, nitrosylated cysteine residues (-SNO) were reduced to free cysteine by incubating 1 h with 30 mM sodium ascorbate and selectively labeled with HPDP-biotin. Proteins were precipitated again with acetone to wash the excess of HPDP-biotin and solubilized with HENS buffer (HEN buffer with 1% SDS). Following solubilization, the samples were incubated 1 h with agarose-conjugated streptavidin beads (Sigma-Aldrich Chemical, St Louis, MO, USA) and centrifuged to pull HPDP-biotinylated proteins down. Adsorbed proteins were resolved in a 10% SDS-PAGE, electroblotted to a nitrocellulose membrane and stained with Ponceau red to evaluate the total protein content. Then, the biotinylated proteins were probed with anti-Cx43 (Sigma Aldrich Chemical, St Louis, MO, USA) antibodies. For all Western blot analysis, the intensity of the signal was evaluated using the ImageJ program (NIH public domain software).

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).