connexin 43 cx43 polyclonal rabbit antibodies Search Results


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  • 94
    Thermo Fisher rabbit polyclonal anti connexin43
    HUVEC and cFB acquired the cardiac phenotype through cell fusion with cardiomyocytes. (A) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–d) or GFP + cFB (e–h) and stained with mouse monoclonal anti-cTnT (red) and rabbit <t>polyclonal</t> anti-β-gal antibodies (blue). Merged images were obtained from the same confocal plane. GFP + HUVEC and GFP + cFB (a and e, arrow) expressed cTnT (b and f, arrow) and β-gal (c and g, arrow) in the same cell (merged on d and h). Arrowheads indicate the nonfused cardiomyocytes. Bars, 50 μm. (B) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–f) or GFP + cFB (g–l) and stained with mouse monoclonal anti-cTnT (red) and goat polyclonal anti-GATA4 or rabbit polyclonal anti-ANF or <t>anti-connexin43</t> antibodies (blue). cTnT-expressing GFP + HUVEC (a, c, and e) and cTnT-expressing GFP + cFB (g, i, and k) expressed GATA4 (b and h, arrowhead), ANF (d and j, arrowhead), and connexin43 (f and l, arrowhead). Bars, 50 μm.
    Rabbit Polyclonal Anti Connexin43, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore anti connexin 43 antibody
    Areas with higher connexin 43 (Cx43) expression and mis-localization show calcium overload. Von Kossa staining to detect calcium deposits in heart sections from uninfected and HIV-infected individuals was performed. All these sections correspond to serial sections in which Cx43 was up-regulated or mis-localized. Sections stained for DAPI, Cx43, and lipofuscin (autofluorescence), and the merge, correspond to the same tissue section. The adjacent tissue section was stained for Von Kossa (labeled calcium).  A:  Staining for tissue sections obtained from uninfected individuals (uninfected) showing normal Cx43 distribution and low accumulation of lipofuscin with minimal calcium deposits. Staining for tissue sections obtained from HIV-infected individuals (HIV + ) showing increased expression of Cx43 in several areas of the tissue and increased accumulation of lipofuscin also shows increased accumulation of calcium deposits in vast areas of the tissue (different degrees of brown-back staining).  B:  Quantification of calcium staining (dark staining) indicates an increase of 60% in Von Kossa staining. Data are expressed as means ± SD.  n  = 29 different individuals ( A  and  B ). Scale bar = 200 μm. A.U., arbitrary unit.
    Anti Connexin 43 Antibody, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1769 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti connexin 43 antibody/product/Millipore
    Average 99 stars, based on 1769 article reviews
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    97
    Thermo Fisher rabbit polyclonal anti cx43 antibody
    MMC induces alteration in <t>Cx43</t> association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit <t>polyclonal</t> anti-Cx43 was performed followed by immunoblotting
    Rabbit Polyclonal Anti Cx43 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 127 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal anti cx43 antibody/product/Thermo Fisher
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    99
    Cell Signaling Technology Inc rabbit polyclonal anti connexin 43
    MMC induces alteration in <t>Cx43</t> association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit <t>polyclonal</t> anti-Cx43 was performed followed by immunoblotting
    Rabbit Polyclonal Anti Connexin 43, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    HUVEC and cFB acquired the cardiac phenotype through cell fusion with cardiomyocytes. (A) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–d) or GFP + cFB (e–h) and stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged images were obtained from the same confocal plane. GFP + HUVEC and GFP + cFB (a and e, arrow) expressed cTnT (b and f, arrow) and β-gal (c and g, arrow) in the same cell (merged on d and h). Arrowheads indicate the nonfused cardiomyocytes. Bars, 50 μm. (B) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–f) or GFP + cFB (g–l) and stained with mouse monoclonal anti-cTnT (red) and goat polyclonal anti-GATA4 or rabbit polyclonal anti-ANF or anti-connexin43 antibodies (blue). cTnT-expressing GFP + HUVEC (a, c, and e) and cTnT-expressing GFP + cFB (g, i, and k) expressed GATA4 (b and h, arrowhead), ANF (d and j, arrowhead), and connexin43 (f and l, arrowhead). Bars, 50 μm.

    Journal: The Journal of Cell Biology

    Article Title: Cardiomyocytes fuse with surrounding noncardiomyocytes and reenter the cell cycle

    doi: 10.1083/jcb.200312111

    Figure Lengend Snippet: HUVEC and cFB acquired the cardiac phenotype through cell fusion with cardiomyocytes. (A) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–d) or GFP + cFB (e–h) and stained with mouse monoclonal anti-cTnT (red) and rabbit polyclonal anti-β-gal antibodies (blue). Merged images were obtained from the same confocal plane. GFP + HUVEC and GFP + cFB (a and e, arrow) expressed cTnT (b and f, arrow) and β-gal (c and g, arrow) in the same cell (merged on d and h). Arrowheads indicate the nonfused cardiomyocytes. Bars, 50 μm. (B) LacZ -expressing cardiomyocytes of neonatal rats were cocultured with GFP + HUVEC (a–f) or GFP + cFB (g–l) and stained with mouse monoclonal anti-cTnT (red) and goat polyclonal anti-GATA4 or rabbit polyclonal anti-ANF or anti-connexin43 antibodies (blue). cTnT-expressing GFP + HUVEC (a, c, and e) and cTnT-expressing GFP + cFB (g, i, and k) expressed GATA4 (b and h, arrowhead), ANF (d and j, arrowhead), and connexin43 (f and l, arrowhead). Bars, 50 μm.

    Article Snippet: The following antibodies were used for immunostaining: mouse monoclonal anti-cTnT (RV-C2, DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH), goat polyclonal anti-cTnT, goat polyclonal anti-GATA4 (Santa Cruz Biotechnology, Inc.), rabbit polyclonal anti-ANF (Peninsula Laboratories), rabbit polyclonal anti-connexin43 (Zymed Laboratories), rabbit polyclonal anti-desmin, rabbit polyclonal anti-vWF, mouse monoclonal anti–rat Ki67, mouse monoclonal anti–human Ki67 (Dako Cytomation), mouse monoclonal anti-β-gal, rabbit polyclonal anti-β-gal (CHEMICON International, Inc.), mouse monoclonal anti-vimentin, mouse monoclonal anti-Cre (Sigma-Aldrich), rabbit polyclonal anti-PH3 (Upstate Biotechnology), mouse monoclonal anti-cyclinB1 (Neomarkers), and rabbit polyclonal anti-RFP (MBL International Corporation).

    Techniques: Expressing, Staining

    Areas with higher connexin 43 (Cx43) expression and mis-localization show calcium overload. Von Kossa staining to detect calcium deposits in heart sections from uninfected and HIV-infected individuals was performed. All these sections correspond to serial sections in which Cx43 was up-regulated or mis-localized. Sections stained for DAPI, Cx43, and lipofuscin (autofluorescence), and the merge, correspond to the same tissue section. The adjacent tissue section was stained for Von Kossa (labeled calcium).  A:  Staining for tissue sections obtained from uninfected individuals (uninfected) showing normal Cx43 distribution and low accumulation of lipofuscin with minimal calcium deposits. Staining for tissue sections obtained from HIV-infected individuals (HIV + ) showing increased expression of Cx43 in several areas of the tissue and increased accumulation of lipofuscin also shows increased accumulation of calcium deposits in vast areas of the tissue (different degrees of brown-back staining).  B:  Quantification of calcium staining (dark staining) indicates an increase of 60% in Von Kossa staining. Data are expressed as means ± SD.  n  = 29 different individuals ( A  and  B ). Scale bar = 200 μm. A.U., arbitrary unit.

    Journal: The American Journal of Pathology

    Article Title: HIV-Associated Cardiovascular Disease

    doi: 10.1016/j.ajpath.2017.05.011

    Figure Lengend Snippet: Areas with higher connexin 43 (Cx43) expression and mis-localization show calcium overload. Von Kossa staining to detect calcium deposits in heart sections from uninfected and HIV-infected individuals was performed. All these sections correspond to serial sections in which Cx43 was up-regulated or mis-localized. Sections stained for DAPI, Cx43, and lipofuscin (autofluorescence), and the merge, correspond to the same tissue section. The adjacent tissue section was stained for Von Kossa (labeled calcium). A: Staining for tissue sections obtained from uninfected individuals (uninfected) showing normal Cx43 distribution and low accumulation of lipofuscin with minimal calcium deposits. Staining for tissue sections obtained from HIV-infected individuals (HIV + ) showing increased expression of Cx43 in several areas of the tissue and increased accumulation of lipofuscin also shows increased accumulation of calcium deposits in vast areas of the tissue (different degrees of brown-back staining). B:  Quantification of calcium staining (dark staining) indicates an increase of 60% in Von Kossa staining. Data are expressed as means ± SD. n  = 29 different individuals ( A and B ). Scale bar = 200 μm. A.U., arbitrary unit.

    Article Snippet: Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C.

    Techniques: Expressing, Staining, Infection, Labeling

    Lateralized connexin 43 (Cx43) does not colocalize with N-cadherin in heart tissues obtained from HIV-infected individuals. Postmortem ventricular heart tissue was used. Immunohistochemistry was performed using paraffin-embedded and fresh tissues and analyzed by confocal microscopy and three-dimensional reconstruction to examine colocalization of Cx43 and N-cadherin.  A:  Representative tissue staining for nuclei (DAPI), Cx43 (fluorescein isothiocyanate), and N-cadherin (cyanine 5) in uninfected and HIV-infected tissues.  Arrows  indicate lateralized Cx43 that also lacks staining for N-cadherin. In heart tissues obtained from uninfected individuals, most Cx43 localizes at the intercalated disk. In contrast, in heart tissues obtained from HIV-infected individuals, most Cx43 does not colocalize with N-cadherin and mainly localizes at the lateral membrane of the cardiomyocyte.  B:  Quantification of Cx43-positive pixels that colocalize with N-cadherin–positive pixels in HIV −  (control) and HIV +  conditions. Data are expressed as means ± SD. Scale bar = 38 μm.

    Journal: The American Journal of Pathology

    Article Title: HIV-Associated Cardiovascular Disease

    doi: 10.1016/j.ajpath.2017.05.011

    Figure Lengend Snippet: Lateralized connexin 43 (Cx43) does not colocalize with N-cadherin in heart tissues obtained from HIV-infected individuals. Postmortem ventricular heart tissue was used. Immunohistochemistry was performed using paraffin-embedded and fresh tissues and analyzed by confocal microscopy and three-dimensional reconstruction to examine colocalization of Cx43 and N-cadherin. A: Representative tissue staining for nuclei (DAPI), Cx43 (fluorescein isothiocyanate), and N-cadherin (cyanine 5) in uninfected and HIV-infected tissues. Arrows indicate lateralized Cx43 that also lacks staining for N-cadherin. In heart tissues obtained from uninfected individuals, most Cx43 localizes at the intercalated disk. In contrast, in heart tissues obtained from HIV-infected individuals, most Cx43 does not colocalize with N-cadherin and mainly localizes at the lateral membrane of the cardiomyocyte. B: Quantification of Cx43-positive pixels that colocalize with N-cadherin–positive pixels in HIV − (control) and HIV + conditions. Data are expressed as means ± SD. Scale bar = 38 μm.

    Article Snippet: Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C.

    Techniques: Infection, Immunohistochemistry, Confocal Microscopy, Staining

    HIV infection increases expression of connexin 43 (Cx43) mRNA and protein in human ventricular heart tissue.  A:  Representative quantitative RT-PCR (RT-qPCR) amplifications for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Cx43.  B:  Quantification of the RT-qPCR experiments for tissues from uninfected and HIV-infected hearts. HIV infection (independent of antiretroviral treatment, CD4 counts, replication, and associated inflammation) increases Cx43 expression (mRNA and protein). Each point represents one individual.  C:  Representative Western blot analysis of Cx43 and GAPDH protein expression in HIV −  and HIV +  hearts. GAPDH was used as a loading control; mouse astrocytes were used as a positive control for Cx43 and its phosphorylation (data not shown).  D:  Quantification of the Western blots for heart tissues obtained from uninfected and HIV-infected individuals. Each point represents one individual. Data are expressed as means ± SD. CT, cycle threshold.

    Journal: The American Journal of Pathology

    Article Title: HIV-Associated Cardiovascular Disease

    doi: 10.1016/j.ajpath.2017.05.011

    Figure Lengend Snippet: HIV infection increases expression of connexin 43 (Cx43) mRNA and protein in human ventricular heart tissue. A: Representative quantitative RT-PCR (RT-qPCR) amplifications for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Cx43. B: Quantification of the RT-qPCR experiments for tissues from uninfected and HIV-infected hearts. HIV infection (independent of antiretroviral treatment, CD4 counts, replication, and associated inflammation) increases Cx43 expression (mRNA and protein). Each point represents one individual. C: Representative Western blot analysis of Cx43 and GAPDH protein expression in HIV − and HIV + hearts. GAPDH was used as a loading control; mouse astrocytes were used as a positive control for Cx43 and its phosphorylation (data not shown). D: Quantification of the Western blots for heart tissues obtained from uninfected and HIV-infected individuals. Each point represents one individual. Data are expressed as means ± SD. CT, cycle threshold.

    Article Snippet: Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C.

    Techniques: Infection, Expressing, Quantitative RT-PCR, Western Blot, Positive Control

    HIV infection increases the accumulation of collagen/fibrin in areas with increased expression and mis-localized connexin 43 (Cx43). Trichrome staining (blue) was performed to detect accumulation of collagen and fibrin in serial sections with areas already identified to have increased and mis-localized Cx43 expression.  A:  Trichrome staining of tissue sections obtained from uninfected and HIV-infected individuals.  B:  Quantification of trichrome staining from individuals indicates that HIV infection increases tissue scaring.  n  = 10 uninfected individuals ( A  and  B );  n  = 19 HIV-infected individuals ( A  and  B ). Scale bar = 200 μm. A.U., arbitrary unit. Data are expressed as means ± SD.

    Journal: The American Journal of Pathology

    Article Title: HIV-Associated Cardiovascular Disease

    doi: 10.1016/j.ajpath.2017.05.011

    Figure Lengend Snippet: HIV infection increases the accumulation of collagen/fibrin in areas with increased expression and mis-localized connexin 43 (Cx43). Trichrome staining (blue) was performed to detect accumulation of collagen and fibrin in serial sections with areas already identified to have increased and mis-localized Cx43 expression. A: Trichrome staining of tissue sections obtained from uninfected and HIV-infected individuals. B: Quantification of trichrome staining from individuals indicates that HIV infection increases tissue scaring. n  = 10 uninfected individuals ( A and B ); n  = 19 HIV-infected individuals ( A and B ). Scale bar = 200 μm. A.U., arbitrary unit. Data are expressed as means ± SD.

    Article Snippet: Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C.

    Techniques: Infection, Expressing, Staining

    Electron microscopic analysis of hearts obtained from HIV-infected individuals shows that areas with compromised connexin 43 (Cx43) localization and calcium metabolism have extensive mitochondrial proliferation and decreased myofibril width. Areas with normal and increased Cx43 expression were identified and subjected to electron microscopy analysis.  A  and  B:  Representative sections from an HIV −  heart.  Black lines with circles  indicate myofibril width.  C  and  D:  Representative sections from an HIV +  heart showing mitochondrial proliferation ( arrows ) and a decrease in myofibril width ( black lines with circles ).  E:  Quantification of mitochondrial numbers in heart tissue showing a significant increase in HIV +  tissues.  F:  Quantification of myofibril width in heart tissues obtained from uninfected and HIV-infected individuals. There are no statistically significant differences between uninfected and HIV-infected individuals in myofibril width. However, analysis of tissues obtained from HIV-infected individuals indicates the presence of two distinct populations of myofibrils, one with normal width and a compromised one that is significantly different that that uninfected individual. Data are expressed as means ± SD.  n  = 5 grids per patient from five different individuals were analyzed ( F ).  P  = 0.01, HIV small myofibers compared with uninfected conditions ( F ). Scale bar = 1.9 μm.

    Journal: The American Journal of Pathology

    Article Title: HIV-Associated Cardiovascular Disease

    doi: 10.1016/j.ajpath.2017.05.011

    Figure Lengend Snippet: Electron microscopic analysis of hearts obtained from HIV-infected individuals shows that areas with compromised connexin 43 (Cx43) localization and calcium metabolism have extensive mitochondrial proliferation and decreased myofibril width. Areas with normal and increased Cx43 expression were identified and subjected to electron microscopy analysis. A and B: Representative sections from an HIV − heart. Black lines with circles indicate myofibril width. C and D: Representative sections from an HIV + heart showing mitochondrial proliferation ( arrows ) and a decrease in myofibril width ( black lines with circles ). E: Quantification of mitochondrial numbers in heart tissue showing a significant increase in HIV + tissues. F: Quantification of myofibril width in heart tissues obtained from uninfected and HIV-infected individuals. There are no statistically significant differences between uninfected and HIV-infected individuals in myofibril width. However, analysis of tissues obtained from HIV-infected individuals indicates the presence of two distinct populations of myofibrils, one with normal width and a compromised one that is significantly different that that uninfected individual. Data are expressed as means ± SD. n  = 5 grids per patient from five different individuals were analyzed ( F ). P  = 0.01, HIV small myofibers compared with uninfected conditions ( F ). Scale bar = 1.9 μm.

    Article Snippet: Tissue sections were incubated in blocking solution for 2 hours at room temperature and then in diluted primary antibody (anti-pan Cx43: dilution 1:2000, Sigma C6219; Sigma-Aldrich, St. Louis, MO; or anti–N-cadherin: dilution 1:250, BD 61092; Becton Dickinson, Franklin Lakes, NJ) overnight at 4°C.

    Techniques: Infection, Expressing, Electron Microscopy

    Cloned Sca-1 + CD45 − cells differentiate into cardiac cells in vitro . (A–C) After treatment with 5-azacytidine, TGF-β and vitamin C, cloned Sca-1 + CD45 − cells differentiated in vitro into cardiomyocytes expressing cardiac Troponin T (A), sarcomeric α-actinin + (SA; B), and connexin-43 (C). (D–H) After treatment VEGF, the cloned cells differentiated into endothelial cells expressing CD31 (D), Von Willebrand Factor (VWF; F), and Flk1 (G), and smooth muscle cells expressing SMA (H). The endothelial cells also demonstrated acetylated LDL uptake (Dil-ac-LDL; E). Nuclei were stained with DAPI. Scale bar = 15 µm in A, D, E, Scale bar = 35 µm in B, C, F, G and H. Typical results are shown (N = 3).

    Journal: PLoS ONE

    Article Title: Sca-1+ Cardiosphere-Derived Cells Are Enriched for Isl1-Expressing Cardiac Precursors and Improve Cardiac Function after Myocardial Injury

    doi: 10.1371/journal.pone.0030329

    Figure Lengend Snippet: Cloned Sca-1 + CD45 − cells differentiate into cardiac cells in vitro . (A–C) After treatment with 5-azacytidine, TGF-β and vitamin C, cloned Sca-1 + CD45 − cells differentiated in vitro into cardiomyocytes expressing cardiac Troponin T (A), sarcomeric α-actinin + (SA; B), and connexin-43 (C). (D–H) After treatment VEGF, the cloned cells differentiated into endothelial cells expressing CD31 (D), Von Willebrand Factor (VWF; F), and Flk1 (G), and smooth muscle cells expressing SMA (H). The endothelial cells also demonstrated acetylated LDL uptake (Dil-ac-LDL; E). Nuclei were stained with DAPI. Scale bar = 15 µm in A, D, E, Scale bar = 35 µm in B, C, F, G and H. Typical results are shown (N = 3).

    Article Snippet: The cells were incubated with the following primary antibodies diluted in Dako antibody diluent at 40 C overnight: rabbit anti-Nkx2-5, GATA4 (Santa Cruz Biotechnology, Santa Cruz, CA), mouse anti-Isl-1 (39.4D5) (Developmental Studies Hybridoma Bank, Iowa City, IA), mouse anti-α SMA (Sigma), mouse anti-Troponin T (Thermo Fisher Scientific, Fremont, CA), mouse anti-SA (Abcam, Cambridge, MA), rabbit anti-connexin-43 (Sigma), mouse anti-CD31 (Abcam) and rabbit anti-VWF (Abcam).

    Techniques: Clone Assay, In Vitro, Expressing, Staining

    αCT1 interacts with Zonula Occludens-1 (ZO-1) PDZ2 and the Connexin 43 (Cx43) Carboxyl Terminus (CT). A)  Schematics of full length Cx43 and αCT1 peptide.  B)  αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain.  C)  Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1.  D)  Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

    Journal: bioRxiv

    Article Title: Targeting the Cx43 Carboxyl Terminal H2 Domain Preserves Left Ventricular Function Following Ischemia-Reperfusion Injury

    doi: 10.1101/668509

    Figure Lengend Snippet: αCT1 interacts with Zonula Occludens-1 (ZO-1) PDZ2 and the Connexin 43 (Cx43) Carboxyl Terminus (CT). A) Schematics of full length Cx43 and αCT1 peptide. B) αCT1 interaction with ZO-1 PDZ domains as indicated by EDC zero-length cross-linking to GST fusion PDZ1, PDZ2 and PDZ3 polypeptides and neutravidin labeling of biotin-tagged peptide at concentrations of 5, 25 and 50 μM. The deletion of the CT Isoleucine (I) in αCT1-I renders this peptide incompetent to interact with the ZO-1 PDZ2 domain. C) Coomassie blue gel of EDC cross-linked products of kinase reaction mixtures containing GST-Cx43 CT and PKC-ε, with (αCT1) and without (Vehicle) αCT1. The fainter band above GST-Cx43 bands (indicated by lines) in the αCT1 lanes were cut from gels and analyzed by Tandem Mass Spectrometry (MS/MS). The boxes to right of gel show Cx43 CT peptides identified by MS/MS as being cross-linked to αCT1. D) Tandem mass spectrum of a quintuply charged crosslinked peptide (m/z: 674.1) between Cx43 345-366 (a-chain) and αCT1 peptide through Cx43 K346 and E8 in αCT1 (b-chain). Only the b-and y-sequence specific ions are labeled. Arrow indicates ion (b a5 2+ ) consistent with cross-linkage between Cx43 CT lysine K346 and the glutamic acid (E) residue of αCT1 at position −1.

    Article Snippet: Samples were co-labeled with a rabbit antibody against either connexin43 (Sigma, C6219, 1:250), Dapi and streptavidin conjugated to AlexaFluor 647 (1:4000; ThermoFisher Scientific).

    Techniques: Labeling, Mass Spectrometry, Tandem Mass Spectroscopy, Sequencing

    Oligodendrocyte Cx47 cannot form functional heterotypic channels with Cx32 or Cx26 but does so readily with Cx43 or Cx30. Cells expressing Cx47 ( A-E , green) were plated with cells expressing Cx32 (A, blue), Cx26 (B, blue), an empty vector control (C,

    Journal: Glia

    Article Title: Functional Heterotypic Interactions Between Astrocyte and Oligodendrocyte Connexins

    doi: 10.1002/glia.21073

    Figure Lengend Snippet: Oligodendrocyte Cx47 cannot form functional heterotypic channels with Cx32 or Cx26 but does so readily with Cx43 or Cx30. Cells expressing Cx47 ( A-E , green) were plated with cells expressing Cx32 (A, blue), Cx26 (B, blue), an empty vector control (C,

    Article Snippet: Cells were grown on glass bottom dishes (MatTek, Ashland, MA) or coverslips, fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 10% FBS in PBS containing 0.2% Tween-20, and incubated for 1 hour at room temperature in appropriate combinations of the following primary antibodies: rabbit anti-Cx43 (1:10,000 dilution; Sigma C-6219), mouse anti-Cx43 (1:100, Zymed 13-8300), rabbit anti-Cx47 (1:1000, ), rabbit anti-Cx32 (1:100; Zymed 34-5700), mouse anti-Cx32 (1:100, Zymed 13-8200), rabbit anti-Cx26 (1:50; Zymed 71-0500), mouse anti-Cx26 (1:100, Zymed 33-5800), rabbit anti-Cx30 (1:1000; Zymed 71-2200), or rabbit anti-GFAP(1:250 dilution; Dako, Carpinteria, CA).

    Techniques: Functional Assay, Expressing, Plasmid Preparation

    Oligodendrocyte Cx32 can form functional heterotypic channels with Cx30 or Cx26. Cells expressing Cx32 ( A-D , green) were mixed with cells expressing Cx43 (A, blue), empty vector controls (B, blue), Cx30 (C, blue), or Cx26 (D, blue). Neurobiotin injection

    Journal: Glia

    Article Title: Functional Heterotypic Interactions Between Astrocyte and Oligodendrocyte Connexins

    doi: 10.1002/glia.21073

    Figure Lengend Snippet: Oligodendrocyte Cx32 can form functional heterotypic channels with Cx30 or Cx26. Cells expressing Cx32 ( A-D , green) were mixed with cells expressing Cx43 (A, blue), empty vector controls (B, blue), Cx30 (C, blue), or Cx26 (D, blue). Neurobiotin injection

    Article Snippet: Cells were grown on glass bottom dishes (MatTek, Ashland, MA) or coverslips, fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 10% FBS in PBS containing 0.2% Tween-20, and incubated for 1 hour at room temperature in appropriate combinations of the following primary antibodies: rabbit anti-Cx43 (1:10,000 dilution; Sigma C-6219), mouse anti-Cx43 (1:100, Zymed 13-8300), rabbit anti-Cx47 (1:1000, ), rabbit anti-Cx32 (1:100; Zymed 34-5700), mouse anti-Cx32 (1:100, Zymed 13-8200), rabbit anti-Cx26 (1:50; Zymed 71-0500), mouse anti-Cx26 (1:100, Zymed 33-5800), rabbit anti-Cx30 (1:1000; Zymed 71-2200), or rabbit anti-GFAP(1:250 dilution; Dako, Carpinteria, CA).

    Techniques: Functional Assay, Expressing, Plasmid Preparation, Injection

    Primary astrocytes lacking Cx43 are communication negative. A ) Primary astrocyte cultures were 95% GFAP-positive. B ) RTPCR screen of control astrocytes reveals abundant Cx43 while astrocytes derived from a conditional Cx43KO showed very weak expression

    Journal: Glia

    Article Title: Functional Heterotypic Interactions Between Astrocyte and Oligodendrocyte Connexins

    doi: 10.1002/glia.21073

    Figure Lengend Snippet: Primary astrocytes lacking Cx43 are communication negative. A ) Primary astrocyte cultures were 95% GFAP-positive. B ) RTPCR screen of control astrocytes reveals abundant Cx43 while astrocytes derived from a conditional Cx43KO showed very weak expression

    Article Snippet: Cells were grown on glass bottom dishes (MatTek, Ashland, MA) or coverslips, fixed with 4% paraformaldehyde for 15 min at room temperature, blocked with 10% FBS in PBS containing 0.2% Tween-20, and incubated for 1 hour at room temperature in appropriate combinations of the following primary antibodies: rabbit anti-Cx43 (1:10,000 dilution; Sigma C-6219), mouse anti-Cx43 (1:100, Zymed 13-8300), rabbit anti-Cx47 (1:1000, ), rabbit anti-Cx32 (1:100; Zymed 34-5700), mouse anti-Cx32 (1:100, Zymed 13-8200), rabbit anti-Cx26 (1:50; Zymed 71-0500), mouse anti-Cx26 (1:100, Zymed 33-5800), rabbit anti-Cx30 (1:1000; Zymed 71-2200), or rabbit anti-GFAP(1:250 dilution; Dako, Carpinteria, CA).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Expressing

    Immunofluorescent identification of connexin expression in transfected HeLa cells. HeLa cells expressing Cx43 (A) and Cx26 (B) stained with antibodies to Cx43 and Cx26, respectively, show typical punctate staining of Cx43 and Cx26 gap junction plaques at cell–cell contact areas, as well as Cx43 and Cx26 localization in the cell membranes of single cells. Green labeling represents staining of connexin proteins, while blue shows DAPI staining of cell nuclei. The right panels show bright field images. Scale bar, 10 μm.

    Journal: Frontiers in Pharmacology

    Article Title: Cyclic nucleotide permeability through unopposed connexin hemichannels

    doi: 10.3389/fphar.2013.00075

    Figure Lengend Snippet: Immunofluorescent identification of connexin expression in transfected HeLa cells. HeLa cells expressing Cx43 (A) and Cx26 (B) stained with antibodies to Cx43 and Cx26, respectively, show typical punctate staining of Cx43 and Cx26 gap junction plaques at cell–cell contact areas, as well as Cx43 and Cx26 localization in the cell membranes of single cells. Green labeling represents staining of connexin proteins, while blue shows DAPI staining of cell nuclei. The right panels show bright field images. Scale bar, 10 μm.

    Article Snippet: Commercially available anti-connexin43 (Sigma) and anti-connexin26 (Zymed Labs) antibodies were used for immunostaining.

    Techniques: Expressing, Transfection, Staining, Labeling

    Characterization of CPVT-iPSCs derived CMs. (A) Immunocytochemical stainings of cardiac markers where red represents troponin T, green connexin-43 and blue DAPI-staining for nuclei. Scale bars 200 μm. (B) Representative traces of a control CM showing normal regular Ca 2+ transients and CPVT1 CMs showing abnormalities like multiple peaks, low peaks, irregular phases and oscillations in Ca 2+ handling. (C) Quantification of percentage of CPVT1 and control iPSC CMs exhibiting abnormal Ca 2+ transients at baseline (bl) and during adrenaline perfusion (adr). (D) Frequency and (E) Diastolic level of intracellular Ca 2+ of all CPVT1 and control CMs. Numbers of cells analyzed in C, D, and E, exon 3 del n = 48, P2328S n = 72, T2538R n = 52, L4115F n = 110, Q4201R n = 63, V4653F n = 29, Controls (WT) n = 28. As an exception, number of WT cells analyzed in D, and E, in bl n = 54 and adr n = 27 and number of P2328S cells in bl n = 90 and adr n = 47. Grey bars indicate cells at baseline and black bars during adrenaline perfusion. Error bars, SEM. *P

    Journal: PLoS ONE

    Article Title: Antiarrhythmic Effects of Dantrolene in Patients with Catecholaminergic Polymorphic Ventricular Tachycardia and Replication of the Responses Using iPSC Models

    doi: 10.1371/journal.pone.0125366

    Figure Lengend Snippet: Characterization of CPVT-iPSCs derived CMs. (A) Immunocytochemical stainings of cardiac markers where red represents troponin T, green connexin-43 and blue DAPI-staining for nuclei. Scale bars 200 μm. (B) Representative traces of a control CM showing normal regular Ca 2+ transients and CPVT1 CMs showing abnormalities like multiple peaks, low peaks, irregular phases and oscillations in Ca 2+ handling. (C) Quantification of percentage of CPVT1 and control iPSC CMs exhibiting abnormal Ca 2+ transients at baseline (bl) and during adrenaline perfusion (adr). (D) Frequency and (E) Diastolic level of intracellular Ca 2+ of all CPVT1 and control CMs. Numbers of cells analyzed in C, D, and E, exon 3 del n = 48, P2328S n = 72, T2538R n = 52, L4115F n = 110, Q4201R n = 63, V4653F n = 29, Controls (WT) n = 28. As an exception, number of WT cells analyzed in D, and E, in bl n = 54 and adr n = 27 and number of P2328S cells in bl n = 90 and adr n = 47. Grey bars indicate cells at baseline and black bars during adrenaline perfusion. Error bars, SEM. *P

    Article Snippet: [ ] 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).

    Techniques: Derivative Assay, Staining

    Comparison of the distribution of microtubules in 2D- and 3D-cardiac cultures. Cells were grown for 48 hours, fixed with 4% paraformaldehyde and triple-stained with polyclonal anti-connexin-43 antibody (red), mouse monoclonal anti-α-tubulin antibody (green), and the DNA-specific probe DAPI (blue). Cells were analyzed in a Leica laser scanning confocal microscope. Different optical focal planes of the cells were acquired and projected in order to show both the 2D-cells and the 3D-aggregates. 2D-cells display an extensive microtubular network (arrow, a ), whereas only a few cells in the surface of the aggregates display organized microtubules. An intense labeling of connexin 43 is only seen in the 3D-aggregates ( a ). To highlight the differences between the 2D and 3D-cells, we projected the slices corresponding to the 3D-aggregate only in image ( b ) and the slices corresponding to the 2D-cells only in image ( c ).

    Journal: PLoS ONE

    Article Title: 2D and 3D-Organized Cardiac Cells Shows Differences in Cellular Morphology, Adhesion Junctions, Presence of Myofibrils and Protein Expression

    doi: 10.1371/journal.pone.0038147

    Figure Lengend Snippet: Comparison of the distribution of microtubules in 2D- and 3D-cardiac cultures. Cells were grown for 48 hours, fixed with 4% paraformaldehyde and triple-stained with polyclonal anti-connexin-43 antibody (red), mouse monoclonal anti-α-tubulin antibody (green), and the DNA-specific probe DAPI (blue). Cells were analyzed in a Leica laser scanning confocal microscope. Different optical focal planes of the cells were acquired and projected in order to show both the 2D-cells and the 3D-aggregates. 2D-cells display an extensive microtubular network (arrow, a ), whereas only a few cells in the surface of the aggregates display organized microtubules. An intense labeling of connexin 43 is only seen in the 3D-aggregates ( a ). To highlight the differences between the 2D and 3D-cells, we projected the slices corresponding to the 3D-aggregate only in image ( b ) and the slices corresponding to the 2D-cells only in image ( c ).

    Article Snippet: Mouse monoclonal anti-BrdU, mouse monoclonal anti-α-tubulin (clone DM 1A), mouse monoclonal anti-sarcomeric alpha-actinin (clone EA53), rabbit polyclonal anti-desmin, rabbit polyclonal anti-connexin 43, and rabbit polyclonal anti-pan-cadherin antibodies were purchased from Sigma Chemical Co. (St. Louis, MO, USA).

    Techniques: Staining, Microscopy, Labeling

    (A) A pipette containing 2 mmol/L LY was attached to the cell furthest to the left in whole cell configuration. Epi-fluorescent micrographs were taken at 1, 5, and 10 min after dye injection into the first cell and showed a progressive fluorescence intensity increase in the two adjacent recipient cells. Scale bar: 20 μm. (B) Quantification of cell-to-cell spread of LY. Fluorescence intensity plots versus time for the 5 mM 4-PB HeLa Cx43 cell trial shown in (A) : first (injected) cell (▲), second cell (■), and third cell (•).

    Journal: Frontiers in Pharmacology

    Article Title: The effects of the histone deacetylase inhibitor 4-phenylbutyrate on gap junction conductance and permeability

    doi: 10.3389/fphar.2013.00111

    Figure Lengend Snippet: (A) A pipette containing 2 mmol/L LY was attached to the cell furthest to the left in whole cell configuration. Epi-fluorescent micrographs were taken at 1, 5, and 10 min after dye injection into the first cell and showed a progressive fluorescence intensity increase in the two adjacent recipient cells. Scale bar: 20 μm. (B) Quantification of cell-to-cell spread of LY. Fluorescence intensity plots versus time for the 5 mM 4-PB HeLa Cx43 cell trial shown in (A) : first (injected) cell (▲), second cell (■), and third cell (•).

    Article Snippet: WESTERN BLOT ANALYSIS The gap junction protein Cx43 was detected in cell cultures by Western blot analysis using a commercially available, polyclonal, anti-Cx43 antibody (C 6219, Sigma).

    Techniques: Transferring, Injection, Fluorescence

    Single channel properties of gap junction channels. (A) Single channel currents elicited by bipolar V j pulses ( V 1 and V 2 ) of 40 mV (top I j trace) and 50 mV (middle I j trace) in HeLa Cx43 cell pairs with 4-PB exposure and control conditions, respectively. Continuous lines indicate zero current level. The current histograms (right) revealed 53 pS and 51–52 pS conductance levels, for 4-PB exposed and control cells, respectively. (B) Histograms of single channel conductances for 4-PB treated (left panel) and control (right panel) cells. The smooth curves represent fits of data to Gaussian distributions. For specific data see text.

    Journal: Frontiers in Pharmacology

    Article Title: The effects of the histone deacetylase inhibitor 4-phenylbutyrate on gap junction conductance and permeability

    doi: 10.3389/fphar.2013.00111

    Figure Lengend Snippet: Single channel properties of gap junction channels. (A) Single channel currents elicited by bipolar V j pulses ( V 1 and V 2 ) of 40 mV (top I j trace) and 50 mV (middle I j trace) in HeLa Cx43 cell pairs with 4-PB exposure and control conditions, respectively. Continuous lines indicate zero current level. The current histograms (right) revealed 53 pS and 51–52 pS conductance levels, for 4-PB exposed and control cells, respectively. (B) Histograms of single channel conductances for 4-PB treated (left panel) and control (right panel) cells. The smooth curves represent fits of data to Gaussian distributions. For specific data see text.

    Article Snippet: WESTERN BLOT ANALYSIS The gap junction protein Cx43 was detected in cell cultures by Western blot analysis using a commercially available, polyclonal, anti-Cx43 antibody (C 6219, Sigma).

    Techniques:

    (A) Western blot analysis of HEK-293 (left panel) and HeLa Cx43 (right panel) cells using polyclonal Cx43 antibody. Lines from left to right correspond to 0, 1, 2, and 5 mM 4-PB, respectively. Anti-α tubulin is shown as a reference indicator. (B) Summary of Western blot data from four experiments with HEK-293 control cells and cells exposed to 5 mM 4-PB. Upon exposure to 5 mM 4-PB Cx43 expression increased to 166 ± 11% which was statistically significant ( p = 0.029) in comparison to the control cells (0 mM 4-PB) where Cx43 expression was assumed to be 100%.

    Journal: Frontiers in Pharmacology

    Article Title: The effects of the histone deacetylase inhibitor 4-phenylbutyrate on gap junction conductance and permeability

    doi: 10.3389/fphar.2013.00111

    Figure Lengend Snippet: (A) Western blot analysis of HEK-293 (left panel) and HeLa Cx43 (right panel) cells using polyclonal Cx43 antibody. Lines from left to right correspond to 0, 1, 2, and 5 mM 4-PB, respectively. Anti-α tubulin is shown as a reference indicator. (B) Summary of Western blot data from four experiments with HEK-293 control cells and cells exposed to 5 mM 4-PB. Upon exposure to 5 mM 4-PB Cx43 expression increased to 166 ± 11% which was statistically significant ( p = 0.029) in comparison to the control cells (0 mM 4-PB) where Cx43 expression was assumed to be 100%.

    Article Snippet: WESTERN BLOT ANALYSIS The gap junction protein Cx43 was detected in cell cultures by Western blot analysis using a commercially available, polyclonal, anti-Cx43 antibody (C 6219, Sigma).

    Techniques: Western Blot, Expressing

    Cx43 Knockdown in human synovial fibroblast-like cells reduced the expression of OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with GJA1 targeting siRNAs ( GJA1 ) reduced the gene expression of some OA-associated (A) catabolic genes and (B) inflammatory genes relative to a scrambled siRNA control (SCR) N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Cx43 Knockdown in human synovial fibroblast-like cells reduced the expression of OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with GJA1 targeting siRNAs ( GJA1 ) reduced the gene expression of some OA-associated (A) catabolic genes and (B) inflammatory genes relative to a scrambled siRNA control (SCR) N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Expressing, Quantitative RT-PCR, Transfection

    Transfection with a Cx43 expression construct increased the expression of IL1B , IL6 and PTGS2 mRNA in HIG82 synovial fibroblasts in vitro. By quantitative real time RT-PCR, transient transfection of HIG82 rabbit synovial fibroblasts with increasing concentrations of a rat Cx43 expressing plasmid (pSFFV-Cx43) dose dependently increased the gene expression of IL1B , IL6 and PTGS2 . N = 3. Total DNA was kept constant in all wells by the inclusion of the appropriate amount of an empty vector (pSFFV-neo). Data are shown as means ± standard deviations. Asterisks indicate p-value

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Transfection with a Cx43 expression construct increased the expression of IL1B , IL6 and PTGS2 mRNA in HIG82 synovial fibroblasts in vitro. By quantitative real time RT-PCR, transient transfection of HIG82 rabbit synovial fibroblasts with increasing concentrations of a rat Cx43 expressing plasmid (pSFFV-Cx43) dose dependently increased the gene expression of IL1B , IL6 and PTGS2 . N = 3. Total DNA was kept constant in all wells by the inclusion of the appropriate amount of an empty vector (pSFFV-neo). Data are shown as means ± standard deviations. Asterisks indicate p-value

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Transfection, Expressing, Construct, In Vitro, Quantitative RT-PCR, Plasmid Preparation

    Transfection of human synovial fibroblast-like cells with a Cx43 expression construct increased the expression of OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with a rat Cx43 expressing plasmid (pSFFV-Cx43) significantly increased the gene expression of the OA-associated catabolic genes (A) MMP1 , MMP13 , ADAMTS4 and ADAMTS5 and the inflammatory cytokine genes (B) IL1 , IL6 and PTGS2 . N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Transfection of human synovial fibroblast-like cells with a Cx43 expression construct increased the expression of OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with a rat Cx43 expressing plasmid (pSFFV-Cx43) significantly increased the gene expression of the OA-associated catabolic genes (A) MMP1 , MMP13 , ADAMTS4 and ADAMTS5 and the inflammatory cytokine genes (B) IL1 , IL6 and PTGS2 . N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Transfection, Expressing, Construct, Quantitative RT-PCR, Plasmid Preparation

    Modulation of Cx43 expression affected the NFκB pathway. Immunoblots from whole cell extracts of transiently transfected SW982 cells showed that (A) overexpression of Cx43 enhanced the abundance of phospho-p65 (RelA) subunit of the NFκB complex while (B) knockdown of Cx43 expression had the opposing effect. GAPDH was used as a load control.

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Modulation of Cx43 expression affected the NFκB pathway. Immunoblots from whole cell extracts of transiently transfected SW982 cells showed that (A) overexpression of Cx43 enhanced the abundance of phospho-p65 (RelA) subunit of the NFκB complex while (B) knockdown of Cx43 expression had the opposing effect. GAPDH was used as a load control.

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Expressing, Western Blot, Transfection, Over Expression

    Transfection with a Cx43 expression construct increased the expression of MMP1 , MMP13 , ADAMTS4 , ADAMTS5 mRNA and MMP activity in HIG82 synovial fibroblasts in vitro. By quantitative real time RT-PCR, transient transfection of HIG82 rabbit synovial fibroblasts with increasing concentrations of a rat Cx43 expressing plasmid (pSFFV-Cx43) dose dependently increased the gene expression of (A) MMP1 , MMP13 as well as the rat Gja1 transgene (please note, these primers do not detect the endogenous rabbit Cx43). N = 3. (B) Likewise, the gene expression of the aggrecanases, ADAMTS4 and ADAMTS5 , were increased by the overexpression of Cx43. N = 3. (C) Matrix metalloproteinase activity in conditioned media from Cx43 transfected HIG82 cells was increased nearly 2-fold relative to cells transfected with an empty vector. N = 3. Total DNA was kept constant in all wells by the inclusion of the appropriate amount of an empty vector (pSFFV-neo). Data are shown as means ± standard deviations. Asterisks indicate p-value

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Transfection with a Cx43 expression construct increased the expression of MMP1 , MMP13 , ADAMTS4 , ADAMTS5 mRNA and MMP activity in HIG82 synovial fibroblasts in vitro. By quantitative real time RT-PCR, transient transfection of HIG82 rabbit synovial fibroblasts with increasing concentrations of a rat Cx43 expressing plasmid (pSFFV-Cx43) dose dependently increased the gene expression of (A) MMP1 , MMP13 as well as the rat Gja1 transgene (please note, these primers do not detect the endogenous rabbit Cx43). N = 3. (B) Likewise, the gene expression of the aggrecanases, ADAMTS4 and ADAMTS5 , were increased by the overexpression of Cx43. N = 3. (C) Matrix metalloproteinase activity in conditioned media from Cx43 transfected HIG82 cells was increased nearly 2-fold relative to cells transfected with an empty vector. N = 3. Total DNA was kept constant in all wells by the inclusion of the appropriate amount of an empty vector (pSFFV-neo). Data are shown as means ± standard deviations. Asterisks indicate p-value

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Transfection, Expressing, Construct, Activity Assay, In Vitro, Quantitative RT-PCR, Plasmid Preparation, Over Expression

    Inhibition of the NFκB pathway prevented the ability of Cx43 overexpression to enhance the expression of some OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with a rat Cx43 expressing plasmid (pSFFV-Cx43) significantly increased the gene expression of the OA-associated catabolic and inflammatory genes (A) MMP1 , ADAMTS5, IL1 and PTGS2 . This effect of Cx43 on the expression of these genes was abrogated by inhibition of the NFκB pathway with MG132 (50 μM, 5 hours). N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Journal: BMC Musculoskeletal Disorders

    Article Title: Connexin43 enhances the expression of osteoarthritis-associated genes in synovial fibroblasts in culture

    doi: 10.1186/1471-2474-15-425

    Figure Lengend Snippet: Inhibition of the NFκB pathway prevented the ability of Cx43 overexpression to enhance the expression of some OA-associated catabolic and inflammatory genes. By quantitative real time RT-PCR, transient transfection of SW982 human synovial fibroblast-like cells with a rat Cx43 expressing plasmid (pSFFV-Cx43) significantly increased the gene expression of the OA-associated catabolic and inflammatory genes (A) MMP1 , ADAMTS5, IL1 and PTGS2 . This effect of Cx43 on the expression of these genes was abrogated by inhibition of the NFκB pathway with MG132 (50 μM, 5 hours). N = 3. Data are shown as means ± standard deviations. Asterisks indicate p-value

    Article Snippet: The rabbit anti-connexin43 antibody was purchased from Sigma.

    Techniques: Inhibition, Over Expression, Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation

    rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi -infected mice. ( A ) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 10 8 plaque-forming units (PFU) of rAdCtrl or a mixture of 10 8 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. ( B ) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). ( C ) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). ( D ) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi -infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. ( E ) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi -infected mice pre- and post-therapy with rAdVax. ( F ) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi -infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P

    Journal: PLoS Pathogens

    Article Title: A Human Type 5 Adenovirus-Based Trypanosoma cruzi Therapeutic Vaccine Re-programs Immune Response and Reverses Chronic Cardiomyopathy

    doi: 10.1371/journal.ppat.1004594

    Figure Lengend Snippet: rAdVax immunotherapy recovered the injured heart tissue of chronically T. cruzi -infected mice. ( A ) Chronically Colombian T. cruzi strain-infected mice were evaluated for heart injury markers pre-therapy (120 dpi) or primed-boosted with 2 × 10 8 plaque-forming units (PFU) of rAdCtrl or a mixture of 10 8 PFU of each adenovirus vaccine preparation (rAdASP2+rAdTS; rAdVax). Mortality was recorded until 230 dpi (110 days post-therapy; dpt), when the surviving mice were analyzed for heart injury markers. ( B ) Kaplan-Meier curve representing the percentages of surviving mice (14–20 mice/group in two independent experiments). ( C ) Relative spleen weight (mg of spleen/g of body) and quantitative immunohistochemical staining (IHS) data for T. cruzi parasitism (nests/100 microscopic fields) in the heart tissue of chronically infected mice (120 and 230 dpi, respectively, pre- and post-therapy). ( D ) IHS showing fibronectin (FN)-stained areas in representative cardiac tissue sections of noninfected (NI) controls and chronically T. cruzi -infected mice pre- (120 dpi) and post-therapy (230 dpi; 110 dpt) with rAdVax. ( E ) Quantification of the FN-stained area (%) and connexin 43 (Cx43)-containing gap junction distances detected using IHS staining of heart tissue sections of NI controls or T. cruzi -infected mice pre- and post-therapy with rAdVax. ( F ) Evaluation of CK-MB activity in the serum of NI controls and T. cruzi -infected mice pre- and post-therapy with rAdVax. The data are presented as the means ± SD. ** P

    Article Snippet: Other antibodies included an anti-F4/80 polyclonal antibody (Caltag, USA), polyclonal rabbit anti-connexin 43 (Cx43) (Sigma, USA), polyclonal rabbit anti-mouse fibronectin (FN) (Gibco-BRL, USA), biotinylated anti-rat immunoglobulin (Dako, Denmark) and biotinylated anti-rabbit immunoglobulin and peroxidase-streptavidin complex (Amersham, UK).

    Techniques: Infection, Mouse Assay, Immunohistochemistry, Staining, Activity Assay

    Hypothetical model of ERΔ7 regulation of GJAI in preterm and term labor. During pregnancy, lower E2 levels are permissive for relatively high expression of hnRNPG, which in turn results in increased ERΔ7 isoform generation by promoting exon 7 exclusion. High levels of ERΔ7 block the action of ERα66/ERα46 resulting in down-regulation of GJA1 and maintaining quiescence. Near term increasing levels of circulating E2 result in down-regulation of hnRNPG that manifest in a decline in ERΔ7 levels thus removing the barrier to ERα66/ERα46 transcriptional activity. This results in an upregulation of GJA1 expression leading to increased uterine contractility and labor.

    Journal: EBioMedicine

    Article Title: Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy

    doi: 10.1016/j.ebiom.2018.11.038

    Figure Lengend Snippet: Hypothetical model of ERΔ7 regulation of GJAI in preterm and term labor. During pregnancy, lower E2 levels are permissive for relatively high expression of hnRNPG, which in turn results in increased ERΔ7 isoform generation by promoting exon 7 exclusion. High levels of ERΔ7 block the action of ERα66/ERα46 resulting in down-regulation of GJA1 and maintaining quiescence. Near term increasing levels of circulating E2 result in down-regulation of hnRNPG that manifest in a decline in ERΔ7 levels thus removing the barrier to ERα66/ERα46 transcriptional activity. This results in an upregulation of GJA1 expression leading to increased uterine contractility and labor.

    Article Snippet: Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20, and then probed with the following primary antibodies: anti- ERα HC-20 (1:200; sc-543; carboxy terminus), anti-ERα G-20 (1:250; sc-544; hinge region), anti-ERα H-184 (1:250; sc-7207; amino terminus) anti-hnRNPG (RBMX) (1:500; sc-48,796) from Santa Cruz Biotechnology, anti-ERα D8H8 (1:1000; #8644; carboxy terminus), anti-ERα D6R2W (1:1000; #13258; amino-terminus) from Cell Signaling Technology, anti-ERα (1:100; ab16660; carboxy terminus) from Abcam and GJA1 (1:1000; C6219) from Sigma-Aldrich.

    Techniques: Expressing, Blocking Assay, Activity Assay

    Expression of ERα isoforms and GJA1 in pregnant human myometrium. Representative western blot (A) and densitometric analysis (B) demonstrate ERΔ7 protein in nuclear (Nuc) extracts significantly declined in term laboring (TL) and term non-laboring (TNL) myometrium compared to myometrium from preterm non-laboring (PTNL) women. (C) ERΔ7 mRNA levels are also down-regulated in the TNL samples as compared to PTNL. Representative western blot (D) and densitometric analysis demonstrated no significant change in ERα66 levels in cytoplasmic (Cyto) (E) and nuclear (F) fractions of TL and TNL myometrium as compared to PTNL. Representative western blot (G) and densitometric analysis (H) demonstrated cytoplasmic GJA1 expression levels were significantly upregulated in the TL and TNL as compared to PTNL myometrium. An increasing trend in GJA1 mRNA levels (I) was also observed towards term. GAPDH and NCOA3 are cytoplasmic and nuclear loading controls. Gene expression was normalized to Rplp0 . *p

    Journal: EBioMedicine

    Article Title: Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy

    doi: 10.1016/j.ebiom.2018.11.038

    Figure Lengend Snippet: Expression of ERα isoforms and GJA1 in pregnant human myometrium. Representative western blot (A) and densitometric analysis (B) demonstrate ERΔ7 protein in nuclear (Nuc) extracts significantly declined in term laboring (TL) and term non-laboring (TNL) myometrium compared to myometrium from preterm non-laboring (PTNL) women. (C) ERΔ7 mRNA levels are also down-regulated in the TNL samples as compared to PTNL. Representative western blot (D) and densitometric analysis demonstrated no significant change in ERα66 levels in cytoplasmic (Cyto) (E) and nuclear (F) fractions of TL and TNL myometrium as compared to PTNL. Representative western blot (G) and densitometric analysis (H) demonstrated cytoplasmic GJA1 expression levels were significantly upregulated in the TL and TNL as compared to PTNL myometrium. An increasing trend in GJA1 mRNA levels (I) was also observed towards term. GAPDH and NCOA3 are cytoplasmic and nuclear loading controls. Gene expression was normalized to Rplp0 . *p

    Article Snippet: Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20, and then probed with the following primary antibodies: anti- ERα HC-20 (1:200; sc-543; carboxy terminus), anti-ERα G-20 (1:250; sc-544; hinge region), anti-ERα H-184 (1:250; sc-7207; amino terminus) anti-hnRNPG (RBMX) (1:500; sc-48,796) from Santa Cruz Biotechnology, anti-ERα D8H8 (1:1000; #8644; carboxy terminus), anti-ERα D6R2W (1:1000; #13258; amino-terminus) from Cell Signaling Technology, anti-ERα (1:100; ab16660; carboxy terminus) from Abcam and GJA1 (1:1000; C6219) from Sigma-Aldrich.

    Techniques: Expressing, Western Blot

    ERΔ7 over-expression inhibits GJA1 expression and disrupts the uterine myocyte contractile ability. ERΔ7 was transiently over-expressed in hTERT-HM Tet3G cells using a doxycycline (Dox; 500 ng/ml) inducible recombinant lentivirus for 48 h in the presence or absence of E2 (100 nM). Representative western blot (A) and densitometric analysis (B) demonstrate a decrease in the protein level of GJA1 upon ERΔ7 overexpression in the presence and absence of E2. Western blot analysis (A) also demonstrates E2 mediated nuclear translocation of ERα66 and ERα46, though no changes in ERα66 and ERα46 levels were found to be associated with ERΔ7 overexpression. Immunocytochemical analysis (C) of hTERT-HM Tet3G cells indicates GJA1 was largely limited to the perinuclear area and cell membrane in control and E2 treated uterine myocytes and was diminished upon ERΔ7 overexpression (Dox) in the presence and absence of E2. (D) Collagen gel contraction analysis reveals reduced contractile responsiveness in ERΔ7 overexpressing hTERT-HM Tet3G cells both in the absence or presence of E2 (100 nM) compared to control, as a result of decreased GJA1 levels. GAPDH and NCOA3 are cytoplasmic and nuclear loading controls. A representative gel with mean gel area (cm 2 ) is given for each group (n = 4), each experiment was performed in triplicate. *p

    Journal: EBioMedicine

    Article Title: Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy

    doi: 10.1016/j.ebiom.2018.11.038

    Figure Lengend Snippet: ERΔ7 over-expression inhibits GJA1 expression and disrupts the uterine myocyte contractile ability. ERΔ7 was transiently over-expressed in hTERT-HM Tet3G cells using a doxycycline (Dox; 500 ng/ml) inducible recombinant lentivirus for 48 h in the presence or absence of E2 (100 nM). Representative western blot (A) and densitometric analysis (B) demonstrate a decrease in the protein level of GJA1 upon ERΔ7 overexpression in the presence and absence of E2. Western blot analysis (A) also demonstrates E2 mediated nuclear translocation of ERα66 and ERα46, though no changes in ERα66 and ERα46 levels were found to be associated with ERΔ7 overexpression. Immunocytochemical analysis (C) of hTERT-HM Tet3G cells indicates GJA1 was largely limited to the perinuclear area and cell membrane in control and E2 treated uterine myocytes and was diminished upon ERΔ7 overexpression (Dox) in the presence and absence of E2. (D) Collagen gel contraction analysis reveals reduced contractile responsiveness in ERΔ7 overexpressing hTERT-HM Tet3G cells both in the absence or presence of E2 (100 nM) compared to control, as a result of decreased GJA1 levels. GAPDH and NCOA3 are cytoplasmic and nuclear loading controls. A representative gel with mean gel area (cm 2 ) is given for each group (n = 4), each experiment was performed in triplicate. *p

    Article Snippet: Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20, and then probed with the following primary antibodies: anti- ERα HC-20 (1:200; sc-543; carboxy terminus), anti-ERα G-20 (1:250; sc-544; hinge region), anti-ERα H-184 (1:250; sc-7207; amino terminus) anti-hnRNPG (RBMX) (1:500; sc-48,796) from Santa Cruz Biotechnology, anti-ERα D8H8 (1:1000; #8644; carboxy terminus), anti-ERα D6R2W (1:1000; #13258; amino-terminus) from Cell Signaling Technology, anti-ERα (1:100; ab16660; carboxy terminus) from Abcam and GJA1 (1:1000; C6219) from Sigma-Aldrich.

    Techniques: Over Expression, Expressing, Recombinant, Western Blot, Translocation Assay

    ERα regulation of GJA1 in hTERT-HM cells. RNA and protein extracts were isolated after 96 h of infection with ERα shRNA lentivirus that targets the 3′UTR of ERα mRNA. Representative western blot (A) and densitometric analysis demonstrate a decline in the nuclear ERα66 (B), ERΔ7 isoforms (C) and ERα46 isoforms (D) and the cytoplasmic GJA1 (E) in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. A significant decline in ERα66/ERα46 (F) and ERΔ7 isoform (G) mRNA levels were also observed in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. GAPDH and Histone H3 are cytoplasmic and nuclear loading controls. Gene expression was normalized to Rplp0 . *p

    Journal: EBioMedicine

    Article Title: Estrogen receptor alpha isoform ERdelta7 in myometrium modulates uterine quiescence during pregnancy

    doi: 10.1016/j.ebiom.2018.11.038

    Figure Lengend Snippet: ERα regulation of GJA1 in hTERT-HM cells. RNA and protein extracts were isolated after 96 h of infection with ERα shRNA lentivirus that targets the 3′UTR of ERα mRNA. Representative western blot (A) and densitometric analysis demonstrate a decline in the nuclear ERα66 (B), ERΔ7 isoforms (C) and ERα46 isoforms (D) and the cytoplasmic GJA1 (E) in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. A significant decline in ERα66/ERα46 (F) and ERΔ7 isoform (G) mRNA levels were also observed in the hTERT-HM cells upon lentivirus-mediated knockdown of ERα compared to non-silencing pGIPZ shRNA lentiviral control. GAPDH and Histone H3 are cytoplasmic and nuclear loading controls. Gene expression was normalized to Rplp0 . *p

    Article Snippet: Blots were blocked with 5% nonfat dry milk in Tris-saline buffer (pH 7.4) containing 0.1% Tween-20, and then probed with the following primary antibodies: anti- ERα HC-20 (1:200; sc-543; carboxy terminus), anti-ERα G-20 (1:250; sc-544; hinge region), anti-ERα H-184 (1:250; sc-7207; amino terminus) anti-hnRNPG (RBMX) (1:500; sc-48,796) from Santa Cruz Biotechnology, anti-ERα D8H8 (1:1000; #8644; carboxy terminus), anti-ERα D6R2W (1:1000; #13258; amino-terminus) from Cell Signaling Technology, anti-ERα (1:100; ab16660; carboxy terminus) from Abcam and GJA1 (1:1000; C6219) from Sigma-Aldrich.

    Techniques: Isolation, Infection, shRNA, Western Blot, Expressing

    Induction of Cx43 by inhibition of IPDE3 and IPDE4. Mesangial cells were treated with IPDE3, IPDE4, IPDE3 plus IPDE4, IBMX or IPDE2 plus IPDE5 for 24 h and subjected to Western blot analysis of Cx43 (a). Densitometric analysis of the data is shown

    Journal: British Journal of Pharmacology

    Article Title: Profiling of functional phosphodiesterase in mesangial cells using a CRE-SEAP-based reporting system

    doi: 10.1038/sj.bjp.0706785

    Figure Lengend Snippet: Induction of Cx43 by inhibition of IPDE3 and IPDE4. Mesangial cells were treated with IPDE3, IPDE4, IPDE3 plus IPDE4, IBMX or IPDE2 plus IPDE5 for 24 h and subjected to Western blot analysis of Cx43 (a). Densitometric analysis of the data is shown

    Article Snippet: After blocking with 3% bovine serum albumin in phosphate-buffered saline (PBS), the membranes were incubated with anti-vasodilator-stimulated phosphoprotein (VASP) antibody (dilution 1 : 1000; Chemicon International, Temecula, CA, U.S.A.), anti-connexin43 (Cx43) antibody (dilution 1 : 2000; Sigma), anti-caspase-3 antibody (dilution 1 : 1000; Cell Signaling, Beverly, MA, U.S.A.), or anti-phospho-mitogen-activated protein (MAP) kinase antibody (dilution 1 : 1000; Cell Signaling).

    Techniques: Inhibition, Western Blot

    MMC induces alteration in Cx43 association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit polyclonal anti-Cx43 was performed followed by immunoblotting

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Rapid Changes in Connexin-43 in Response to Genotoxic Stress Stabilize Cell-Cell Communication in Corneal Endothelium

    doi: 10.1167/iovs.11-7272

    Figure Lengend Snippet: MMC induces alteration in Cx43 association with ZO-1. Cells were treated with prewarmed media containing 5 μM MMC for 60 minutes or media alone (CTRL). ( A ) Coimmunoprecipitation with a rabbit polyclonal anti-Cx43 was performed followed by immunoblotting

    Article Snippet: The beads were discarded and then cleared lysates were incubated with 1 μg antibody per 500 μg of lysate using a rabbit polyclonal anti-Cx43 antibody (Invitrogen) overnight at 4°C.

    Techniques:

    Functional coupling of CMPCs to cardiomyocytes. ( A , B ) Immunolabeling of connexin proteins Cx40 and Cx43. Scale bars represent 30 and 25 µm, respectively. ( C ) Fluorescent microscopy of dye transfer from CMPCs labeled with DiI (red) and Calcein (green) to unlabeled cardiomyocytes. Cardiomyocytes that have imported Calcein are indicated by white arrows. Scale bar represents 45 µm. ( D ) Immunolabeling of GFP (green) and HCN4 (red) in CMPCs transduced with LV-HCN4-GFP. Yellow indicates co-staining. Nuclei were counterstained blue with DAPI. Non-transduced CMPCs, shown in the inset, stained negative for GFP and HCN4. Scale bar represents 45 µm. ( E ) Average beating rates of neonatal rat ventricular myocyte (NRVM) monolayers co-cultured with CMPCs expressing GFP alone (grey) and HCN4 and GFP (red). * indicates P

    Journal: Molecules

    Article Title: Cardiomyocyte Progenitor Cells as a Functional Gene Delivery Vehicle for Long-Term Biological Pacing

    doi: 10.3390/molecules24010181

    Figure Lengend Snippet: Functional coupling of CMPCs to cardiomyocytes. ( A , B ) Immunolabeling of connexin proteins Cx40 and Cx43. Scale bars represent 30 and 25 µm, respectively. ( C ) Fluorescent microscopy of dye transfer from CMPCs labeled with DiI (red) and Calcein (green) to unlabeled cardiomyocytes. Cardiomyocytes that have imported Calcein are indicated by white arrows. Scale bar represents 45 µm. ( D ) Immunolabeling of GFP (green) and HCN4 (red) in CMPCs transduced with LV-HCN4-GFP. Yellow indicates co-staining. Nuclei were counterstained blue with DAPI. Non-transduced CMPCs, shown in the inset, stained negative for GFP and HCN4. Scale bar represents 45 µm. ( E ) Average beating rates of neonatal rat ventricular myocyte (NRVM) monolayers co-cultured with CMPCs expressing GFP alone (grey) and HCN4 and GFP (red). * indicates P

    Article Snippet: The antibodies used recognized Cx40 (Rb polyclonal, Chemicon, Burlington, MA, USA, cat. no. AB1726) or Cx43 (Rb polyclonal, Zymed, Wien, Austria, cat. no. 71-0700).

    Techniques: Functional Assay, Immunolabeling, Microscopy, Labeling, Transduction, Staining, Cell Culture, Expressing

    Expression of Connexin 43 in OECs. A, Immunohistochemistry in an OB section of S100-GFP mice (p26). Low magnification of the OB layers indicating the locations where the images were acquired (square). Nuclei were stained with DRAQ5. A′-A‴, Clusters of Cx43 immunoreactivity (red) colocalize with GFP-labeled fine projections of OECs (arrowheads). Insets: higher magnifications of the area inside the dashed square; Scale bar = 5 μm. B, Average intensity of Cx43 immunoreactivity in each layer of the OB, normalized to intensity in the ONL. Error bars represent SEM. C, Single optical section of a z-stack, with lateral projections showing Cx43 immunoreactivity in a tissue slice with a LY-filled OEC. Inset: higher magnification of the dashed rectangle indicated at the right. Arrowhead: a Cx43 cluster colocalizes with an OEC fine projection. D, Isosurface rendering of the green and red channels of a z-stack similar to the one in C . The red surface was edited to eliminate the clusters not apposed to the cell. Arrowhead: contour surface of the nucleus generated with the blue channel. E, Electron micrograph of the ONL. Glial processes were contoured (blue); red lines: gap junction specializations. Inset: high magnification of the gap junction enclosed in the rectangle.

    Journal: Glia

    Article Title: Olfactory ensheathing cell membrane properties are shaped by connectivity

    doi: 10.1002/glia.20953

    Figure Lengend Snippet: Expression of Connexin 43 in OECs. A, Immunohistochemistry in an OB section of S100-GFP mice (p26). Low magnification of the OB layers indicating the locations where the images were acquired (square). Nuclei were stained with DRAQ5. A′-A‴, Clusters of Cx43 immunoreactivity (red) colocalize with GFP-labeled fine projections of OECs (arrowheads). Insets: higher magnifications of the area inside the dashed square; Scale bar = 5 μm. B, Average intensity of Cx43 immunoreactivity in each layer of the OB, normalized to intensity in the ONL. Error bars represent SEM. C, Single optical section of a z-stack, with lateral projections showing Cx43 immunoreactivity in a tissue slice with a LY-filled OEC. Inset: higher magnification of the dashed rectangle indicated at the right. Arrowhead: a Cx43 cluster colocalizes with an OEC fine projection. D, Isosurface rendering of the green and red channels of a z-stack similar to the one in C . The red surface was edited to eliminate the clusters not apposed to the cell. Arrowhead: contour surface of the nucleus generated with the blue channel. E, Electron micrograph of the ONL. Glial processes were contoured (blue); red lines: gap junction specializations. Inset: high magnification of the gap junction enclosed in the rectangle.

    Article Snippet: Primary antibodies [rabbit anti-BLBP, AB9558; rat anti-NCAM, MAB310; mouse anti-connexin 32 (Cx32), MAB3069; and rabbit anti-Cx26, AB1717 from Chemicon/Millipore; rabbit anti-Cx43 71-0700 from Zymed/Invitrogen] diluted in blocking buffer were incubated overnight at room temperature.

    Techniques: Expressing, Immunohistochemistry, Mouse Assay, Staining, Labeling, Generated