cell signaling cx43 rabbit  (Cell Signaling Technology Inc)

Journal: International Journal of Molecular Sciences

Article Title: Molecular and Histological Effects of Glyphosate on Testicular Tissue of the Lizard Podarcis siculus

doi: 10.3390/ijms23094850

Figure Lengend Snippet: Immunohistochemical localization (brown areas) of Connexin 43 in testis of P. siculus treated with different concentration of glyphosate. ( A ) Control. The signal is evident along all the Sertoli cells (red arrows). ( B ) Low dose. Positivity is only evident along some Sertoli cells (red arrows). ( C ) High dose. ( A ): reduced immunopositivity is evident only in spots (red arrows). ( A , insert): no signal is present in control sections. Black arrow: rosette-shaped cell aggregates. Scale bars correspond to 15 µm in figure ( A – C ) and 50 µm in figure ( A insert).

Article Snippet: All the antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), except anti-mouse Cx43, which was from Elabscience (Houston, TX, USA).

Techniques: Immunohistochemical staining, Concentration Assay, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43

doi: 10.1161/atvbaha.121.317324

Figure Lengend Snippet: Figure 7. During vessel formation, cells within a PC lineage preferentially express and localize Cx43 (connexin 43) at their endothelial interface. A, Transcript levels of cell-specific connexin isoforms from fluorescence-activated cell sorted (Flk-1-eGFP+ [enhanced green fluorescent protein] and Ng2-DsRed+) subpopulations of undifferentiated, days 7 and 10 differentiated double-reporter embryonic stem cells (DR-ESCs). Medians with confidence limits (n=2). B, Fold difference in Gja1 (gap junction alpha-1; Cx43) in magnetic bead–assisted cell separation (MACS)–sorted NG2+ (neural glial antigen-2) cells vs CD31 (cluster of differentiation 31)/PECAM-1+ (platelet-EC adhesion molecule-1) cells from WT-ESCs via qRT-PCR (quantitative real-time polymerase chain reaction). C, Day 10 differentiated WT- ESCs labeled for PECAM-1 (Ci; green in Civ and Cv), NG2 (Cii; red in Civ and Cv), and Cx43 (Ciii; blue in Civ and Cv). Nuclei, DAPI (4',6-diamidino-2-phenylindole; white in Cv). D, Western blot for Cx43 and GAPDH (housekeeping) from CD31+ and NG2+ cells isolated from day 10 WT-ESC–derived vessels by MACS. Raw immunoblot provided in Figure S10. E, Fold difference in total NG2+ cell Cx43 protein vs CD31/PECAM-1+ cell Cx43 levels, summing intensities for all Cx43 bands (D). F, Fold difference in each Cx43 phospho- isoform from NG2+ cells vs CD31+ cells isolated by MACS. G, Embryonic day (E) 9.5 brain microvessel labeled for endothelial cell (EC) PECAM-1 (Gi; blue in Gv), PC PDGFRβ (platelet-derived growth factor receptor-β; Gii; red in Gv), and Cx43 (Giii; green in Gv). Nuclei, DAPI (Giv; white in Gv). Scale bar, 10 μm. Cx43 GJ plaques at the cell-cell interface (dashed white ovals). H, E14.5 skin vessels from a Flk-1-eGFP; Ng2-DsRed embryo, visualizing ECs (Hi; green in Hv) and PCs (Hii; red in Hv), and labeled Cx43 (Hiii; blue in Hv), within GJ plaques at the cell-cell interface (dashed white oval). Nuclei, DAPI (Hiv, white in Hv). Scale bar, 5 μm (Figure S9). I, P7 mouse retina microvessels labeled for EC PECAM-1 (Ii; blue in Iv), Ng2-DsRed+ cells (Iii; red in Iv), and Cx43 (Iiii; green in Iv). Nuclei, DAPI (Iiv; white in Iv). Scale bar, 10 μm. Cx43 GJ plaques at cell-cell interface (yellow arrowheads). TPM, Transcripts per million

Article Snippet: Membranes were exposed (in TBST [tris-buffered saline-tween20]) to primary antibodies rabbit anti-mouse phospho-Cx43 (Cell Signaling; catalog No. 3511) at 1:1000 and goat anti-GAPDH (Abcam; catalog No. ab9485) at 1:1000 overnight at 4 °C and to secondary antibodies donkey anti-rabbit AlexaFluor 488 (Jackson ImmunoResearch; catalog No. 711-545-152) and donkey anti-goat AlexaFluor 647 at 1:10 000 (Jackson ImmunoResearch; catalog No. 705- 605-147) for 2 hours at room temperature.

Techniques: Fluorescence, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Labeling, Western Blot, Isolation, Derivative Assay

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Pericyte Progenitor Coupling to the Emerging Endothelium During Vasculogenesis via Connexin 43

doi: 10.1161/atvbaha.121.317324

Figure Lengend Snippet: Figure 8. Conditional deletion of Cx43/Gja1 (connexin 43/gap junction alpha-1) in Ng2+ pericytes induces variable vascular dysmorphogenesis and pericyte investment. A, Embryonic day (E) 9 of Ng2Cre/+; Cx43+/+ and Ng2Cre/+; Cx43lox/lox mouse major vessels (location denoted within whole embryo, yellow box inside insert) labeled for EC PECAM-1 (platelet-EC adhesion molecule-1). Scale bar, 200 μm. Abnormal vessel morphology (dashed red ovals). B, Graph of genotype distribution from Ng2Cre/+; Cx43lox/+×Cx43lox/lox animal crosses as a percentage of total animals generated. (Continued )

Article Snippet: Membranes were exposed (in TBST [tris-buffered saline-tween20]) to primary antibodies rabbit anti-mouse phospho-Cx43 (Cell Signaling; catalog No. 3511) at 1:1000 and goat anti-GAPDH (Abcam; catalog No. ab9485) at 1:1000 overnight at 4 °C and to secondary antibodies donkey anti-rabbit AlexaFluor 488 (Jackson ImmunoResearch; catalog No. 711-545-152) and donkey anti-goat AlexaFluor 647 at 1:10 000 (Jackson ImmunoResearch; catalog No. 705- 605-147) for 2 hours at room temperature.

Techniques: Labeling, Generated

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: Primers sequences used for SYBR Green assays.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: SYBR Green Assay

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A ): Glial connexins and pannexin1 mRNAs quantification measured by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) in the cortex, hypothalamus and DVC of control mice ( n = 6, 23 and 24 respectively). ** p < 0.01 and *** p < 0.001 compared to cortex. ( B – G ): Confocal images of Connexin-43 (Cx43) (red) and Glial Fibrillary Acidic Protein (GFAP) ( E – G ) or vimentin ( F ) double labeling immunohistochemistry performed within the hypothalamus and Dorsal Vagal Complex (DVC). Scale bar: 200 μm. Inset in F shows Cx43 and vimentin co-localization in α-tanycytes. ( H , I ): Confocal images Cx43 (red) and GFAP (green) double immunofluorescence within the DVC. Cx43 is strongly associated with astrocytes GFAP+ (green) processes. ( J , K ): Confocal images of Cx43 (red) and Ionized Ca 2+ Binding Adapter Molecule 1 (IBA1) (green) double immunofluorescence within the DVC. Note that Cx43 labelling is sometimes found associated with thin processes of IBA + microglia. Scale bars: 20 μm for ( H , J ) and 2 μm for ( I ) and ( K ). AP: area postrema, NTS: nucleus tractus solitarius, cc: central canal; ARC: arcuate nucleus, 3V: third ventricle, ME: median eminence.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Labeling, Immunohistochemistry, Immunofluorescence, Binding Assay

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A , B ): Connexin-43 (Cx43) and Glial Fibrillary Acidic Protein (GFAP) mRNA expression (normalized with Glyceraldehyde-3-phosphate dehydrogenase gene, GAPDH) was assessed by reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) from the hypothalamus ( A ) and dorsal vagal complex (DVC) ( B ) of control ad libitum ( n = 14), 24 h fasted ( n = 13), 24 h fasted plus 2 h refeed ( n = 8) or plus 4 h refeed ( n = 8) mice. ( C ): Representative Western blot analysis of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of control ad libitum ( n = 5) and 24 h fasted ( n = 5) mice. ( D , E ): Cx43 and GFAP mRNA expressions (normalized with GAPDH) were assessed by Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) from the hypothalamus ( D ) and dorsal vagal complex (DVC) ( D ) of Normal Chow (NC) ( n = 9) and High Fat Diet (HFD) fed ( n = 9) mice. ( F ): Representative Western blot analysis and quantification of phosphorylated Cx43 (pS368) and Cx43 expression in the hypothalamus of NC ( n = 9) and HFD-fed ( n = 9) mice. A two-way analysis of variance test was performed between different experimental groups * p < 0.05 and *** p < 0.001 significantly different from control ad libitum. # p < 0.05 and ## p < 0.001 significantly different from 24 h fasted mice. ns: no significant difference.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A ): Confocal images of connexin-43 (Cx43) (red) and bassoon (green) double labeling immunohistochemistry performed within the nucleus tractus solitarius (NTS). Scale bar: 5 μm. Inset: Arrows show Cx43 labelling in close vicinity of synapses labelled by presynaptic bassoon protein. Scale bar 1 µm. ( B ): An example of a voxel (4 × 4 µm) used for the quantification of Cx43/bassoon appositions. Scale bar: 0.5 µm. ( C ): Twelve z-stack images defining the voxel illustrated in B and allowing the identification of Cx43/bassoon appositions. ( D ): Stereological estimation of Cx43/bassoon appositions in the NTS (85.13%) and arcuate nucleus of the hypothalamus (ARC) (80.46%) nuclei. ( E ): Table gathering the different stereological values acquired or estimated for each studied animal.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Labeling, Immunohistochemistry

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A ): Representative images depicting ethidium bromide (EtBr) uptake via hemichannels in hypothalamic (ARC) slices treated with either artificial cerebrospinal fluid (aCSF), 200 μM carbenoxolone (Cbx) or 100 µM the connexin-43 (Cx43) hemichannel blocker TAT-GAP19 for 15 min, then exposed to EtBr for 10 min. After fixation, slices were submitted to Glial Fibrillary Acidic Protein (GFAP) immunostaining and Hoechst labelling. Scale bar: 15 µm. ( B ): Quantification of EtBr fluorescence measured in GFAP+ astrocytes. n = 5, with each n representing the average of three replicates. ** p < 0.01.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Immunostaining, Fluorescence

Journal: Cells

Article Title: Blockade of Glial Connexin 43 Hemichannels Reduces Food Intake

doi: 10.3390/cells9112387

Figure Lengend Snippet: ( A ): Cumulative food intake (g), measured over a 24 h period, of mice having received an i.c.v. injection of either vehicle (Sodium Chloride (NaCl) 0.9%) or TAT-GAP19 (12.5 or 25 µg). The TAT-GAP19-I14A, which includes a point mutation making it ineffective on connexin 43 hemichannels (Cx43 HCs) activity was tested on food intake at the dose of 25 µg. ( B ): Graph showing meal size over 24 h after i.c.v. injection of vehicle or TAT-GAP19 25 µg. Note that each bar represents a meal and its width the meal duration. The dark period is represented by the shaded box. ( C – F ): Meal microstructure analysis performed during the dark period for mice that received either vehicle or TAT-GAP19 25 µg allowed quantification of meals number ( C ), meals size (in mg of food, ( D )), meal duration (in min, ( E )) and interprandial period (in min, average intervals between meals, ( F – G )): Measure of energy expenditure (in Kcal/day-1/kg 0.75 ) within 12 h after injection of 0.9% NaCl vehicle ( n = 7) or 50 µg TAT-GAP19 ( n = 7). ( H , I ): Measure of total spontaneous activity (in Arbitrary Unit, UA; ( H ) and cumulative spontaneous activity ( I ) within 12 h for the 2 groups. ns: no significant difference; * p < 0.05, ** p < 0.01 and *** p < 0.001, significantly different from NaCl-treated mice.

Article Snippet: Anti-connexin 43: The blots were blocked for 30 min with 2% casein in PBS- 0,1% Tween 20 and incubated for 2 h at room temperature with rabbit polyclonal antibody against mouse monoclonal anti-Cx43 antibody (1/13000; Sigma Aldrich, St. Louis, MO, USA).

Techniques: Injection, Mutagenesis, Activity Assay

Journal: Medicine

Article Title: Effect of gap junctions on RAW264.7 macrophages infected with H37Rv

doi: 10.1097/MD.0000000000012125

Figure Lengend Snippet: H37Rv infection induces host macrophage connexin expression. RAW264.7 cells were infected by H37Rv strains for 12 hours. Total RNA and protein were tested using RT-PCR (Cx37 and Cx43 mRNA) (A) and Western blotting (Cx43 protein) (B). Both the control and infection cells were collected and tested using immunofluorescence analysis (C). Data are expressed as mean±SD from 3 independent experiments. ∗ P < .05 versus control; ∗∗ P < .01 versus control.

Article Snippet: [ ] A concentration of rabbit anti-mouse Cx43 primary antibodies (1:1000) was purchased from Cell Signaling Technology (Danvers, MA), and mouse anti-mouse β-actin (1:1000) was purchased from ZSGB-BIO (Beijing, China).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Control, Immunofluorescence

Journal: Genetics and molecular research : GMR

Article Title: Tiaogan Qingxin Granule treatment increases myocardial connexin 43 expression in a rat model of arrhythmia.

doi: 10.4238/gmr.15027934

Figure Lengend Snippet: Figure 2. Western blots of Cx43 and GAPDH proteins in each group. Lane 1, Sham-operated group. Lane 2, Arrhythmia model group. Lane 3, Wenxin Granule group. Lane 4, Tiaogan Qingxin Granule group.

Article Snippet: The proteins were diluted with 0.9% NaCl to obtain same concentration and same amounts were denatured at 100°C for 3 min. Proteins were then separated by 10% sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membranes, stored at 4°C for 80 min, and blocked with 5% TBST in skim milk for 1 h. Subsequently, the membranes were treated with rabbit anti-mouse Cx43 polyclonal primary antibody (diluted 1:1,000 in TBST; Cell Signaling Technology, Danvers, MA, US) at 4°C overnight, washed, and incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:2,000; ZSGB-BIO, Beijing, China) for 1 h at room temperature.

Techniques: Western Blot