Co-localization of IP
R with Cx43 in gap junctions of ventricular myocytes. Neonatal rat (A) and adult mouse ventricle tissues (B), and cultured NRVMs (C) were co-immunostained with anti-Cx43 and anti-pan-IP3R antibodies. Representative confocal images show the subcellular distributions of Cx43 (red), pan-IP3R (green) and their co-localization (yellow) in the interfaces between adjacent myocytes. Three-dimensional reconstructions of a single disc of the end-to-end cells display that IP3R partially co-localized with Cx43 in GJs in ventricles (bottom panels in A and B). The enlarged interfaces 1 and 2 (inset in C) show the overlapped distribution of IP3R and Cx43 in NRVMs. Nucleus was stained with Hoechst 33258 (1 μg/ml). Scale bar: 10 μm. The yellow and white arrows in all panels indicate the enhanced IP3R signal in ventricles, and a fraction of Cx43 that is not associated with IP3R in GJs of connected NRVMs, respectively. Solubilized lysates from homogenized NRVMs (D) and mouse ventricles (E) were subjected to immunoprecipitation with anti-Cx43, anti-pan-IP3R, or anti-IP3R isoform antibodies as indicated. Representative western blots show the co-immunoprecipitated Cx43 or IP3R probed with anti-Cx43 or anti-pan-IP3R antibody. Data in all panels are representatives of 3–5 independent experiments.