Anti connexin 43

Differentiated glioblastoma cells were seeded in 24-well plates (Sarstedt, Nümbrecht, Germany) with a density of 1500 cells/well, on glass coverslips (Langenbrinck GmbH, Emmendingen, Germany). Cells were fixed with 4% paraformaldehyde at room temperature (RT) for 20 min. The cells were then permeabilized with 20% Methanol for 15 min. To block unspecific binding of antibody, cells were then incubated with 20% Bovine serum albumin (BSA) at RT for 60 min. Primary antibody mix (200 μL; rabbit polyclonal anti-connexin-43 (Sigma, St. Louis, MO, USA) and rabbit monoclonal ß-tubulin (Cell signaling, Cambridge, UK), dilution 1:1000) was added to each coverslip containing wells and incubated at 37 °C for 60 min. Incubation with secondary antibodies (goat anti-rabbit Alexa Fluor 488 and Alexa Fluor 647 (Sigma, St. Louis, MO, USA), dilution 1:1000) was performed at RT for 60 min. DAPI Fluoromount-G^® mounting medium (SouthernBiotech, Birmingham, AL, USA) was used for nuclei staining. Images were taken with the use of AX 70 microscope and processed with Zeiss Zen software (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). Fluorescence intensity was calculated as follows: integrated density—(Area of selected cells × mean fluorescence of background).

iPSCs were co-cultured with murine visceral endoderm-like (END-2) cells (Humbrecht Institute, Utrecht, The Netherlands) to differentiate them into spontaneously beating CMs. The beating areas of the cell colonies were mechanically excised and treated with collagenase A (Roche Diagnostics).[28 (link)] Single CMs were immunostained with anti-cardiac-troponin-T (1:1500, Abcam, Cambridge, MA, USA), anti-α-actinin (1:1500, Sigma) and anti-connexin-43 (1:1000, Sigma).

All rats were sacrificed on day 21 of EAM. Total protein was prepared from the ventricle of rats. The cardiac ventricles were lysed in RIPA buffer (Product Code: P0013B, Beyotime, China) in the presence of 1 mmol/L phenylmethanesulfonyl fluoride (PMSF, Solarbio, China) and protein phosphatase inhibitor complex I (Aidlab, China), which contains sodium orthovanadate, sodium fluoride, sodium molybdate, sodium tartrate dihydrate, and imidazole. Protein samples were separated by 10% SDS-PAGE gels and were transferred to PVDF membranes (Millipore, United States). The membranes were blocked by 5% non-fat milk for 1 h and incubated with anti-connexin43 (1:10,000, Product Code: C6219, Sigma), anti-phospho-Cx43 (Ser368) (1:1,000, Product Code: 3511, Cell Signaling), anti-phospho-Cx43 (Ser262) (1:100, Product Code: sc-17219, Santa Cruz), anti-myristoylated alanine-rich C kinase substrate (MARCKS) (phospho S158) antibody (1:5,000, Product Code: ab81295, Abcam), and anti-GAPDH antibody (1:5,000, Epitomics). After rinsed with PBST for 5 times, the membranes were incubated with a goat anti-rabbit antibody (1:10,000, Sigma) for 1 h at room temperature. Immunoblots were developed using ChemiDoc™ Imaging Systems (Bio-Rad). Densitometry of bands was analyzed using ImageJ software (National Institutes of Health, United States).

For histology, aCFs alone or cocultures with 50,000 ESC-CMs and 10,000 aCFs or MSCs were plated onto gelatine-coated (0.1%) coverslips. After 2 days, the preparations were fixed with methanol (−20°C, 5 min). Noteworthy, methanol effectively bleaches eGFP. Cells were rehydrated with D-PBS followed by blocking with Roti-Block (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) for 1 hour. Incubations with anti-sarcomeric-α-actinin (Sigma, clone EA53, 1 : 800), anticonnexin 43 (Sigma, rabbit, polyclonal, C6219, 1 : 400), antivimentin (Sigma, mouse, clone VIM-13.2, IgM, V5255; 1 : 200), and anti-smooth muscle actin (Sigma, mouse, clone 1A4, IgG2a, A2547, 1 : 500) were done overnight at 4°C in 1% BSA in PBS. Secondary antibodies (anti-rabbit-AlexaFluor 488, anti-mouse-IgM-AlexaFluor 555, anti-mouse-IgG1-AlexaFluor 647, and anti-mouse-IgG2a-AlexaFluor 647 (all Life Technologies, 1 : 1000)) were applied for 60 min at room temperature. Nuclei were stained using Hoechst 33342 (Sigma-Aldrich Chemie GmbH, Steinheim, Germany). After washing, samples were embedded in ProLong Gold Antifade Reagent (Life Technologies). Images were acquired using an Axiovert 200 M equipped with the Zeiss ApoTome using Axiovision Release 4.4 (both Carl Zeiss, Jena, Germany).

The cardiac ventricles of the rat and the cultured H9c2 cells (Chinese Academy of Sciences Cell Bank, Shanghai) were homogenized on ice and lysed in RIPA buffer (P0013B Beyotime, China) supplemented with proteinase and phosphatase inhibitors. Each protein in the sample was separated by 10% SDS‐PAGE gels and transferred to the PVDF membrane (Millipore, USA). The membranes were blocked by 5% skim milk in PBS containing 0.05% Tween 20 for 1 hour and incubated with anti‐GAPDH antibody (Abcam, 1:5000), anti‐connexin43 (Sigma, 1:10000), anti‐phospho‐Cx43 (Ser368) (Cell Signalling, 1:1000), anti‐p38 (Cell Signalling, 1:1000) and anti‐phospho‐p38 (Thr180/Tyr182) (Cell Signalling, 1:1000) overnight at 4°C. Afterwards, the membranes were washed in PBS containing 0.05% Tween 20, followed by incubation with horseradish peroxidase‐conjugated secondary antibodies (Jackson, 1:10000) for 1 hour at room temperature. The immunoreactivity was visualized by an enhanced chemiluminescence (ECL) advanced kit (Millipore). Signal intensities were analysed using Image J software.

Protein samples were extracted using RIPA buffer (Thermo Fisher Scientific) and separated using 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE, T-Pro, Taipei City, Taiwan) for western blot analysis. The proteins were then transferred to polyvinylidene difluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The membranes were blocked in 5% nonfat dry milk in 0.5% PBS-T. Immune complexes were formed by incubating the proteins with primary antibodies overnight at 4 °C (anti-ZO-1, Abcam, Waltham, MA, USA; anti-E-cadherin, Elabscience, Houston, TX, USA; anti-human desmoplakin 1&2, EMD Millipore, Burlington, MA, USA; anti-JAM-A, Invitrogen, Waltham, MA, USA; anti-β-catenin, EMD Millipore, USA; anti-connexin-43, Sigma-Aldrich, Saint Louis, MO, USA; anti-β-actin, EMD Millipore, USA). Then, the membranes were washed and incubated for 1 h with goat anti-mouse or rabbit HRP-tagged secondary antibodies (1:5000; Leadgen, Tainan, Taiwan). Immunoreactive protein bands were subsequently detected using a luminescence/fluorescence imaging system (GE Healthcare).