rabbit connexin 43 primary antibody  (Boster Bio)

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 4. Glucocorticoids control endothelial and osteoblasts interactions via Cx43. A) RNA was isolated from 3D devices in the presence of Hyd, Pred, and Psl after 18 days. The expression of cell-cell adhesion genes including zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-cad), and connexin43 (Cx43) was examined by RT-qPCR. The transcript levels were normalized to 18S. N = 3. B) The protein expression of Cx43 in the presence of GCs was analyzed by immunoblotting. The below panel shows the quantification of Cx43 levels compared to control. N = 5. C) Representative images of immunofluorescent signals for Cx43 (green), IB4 (red), and DAPI (blue) in bone sections from a mouse implanted with Psl pellets for 60 days or corresponding placebo control. Scale bars, 20 μm and 5 μm (zoom in images); Red arrows indicate the presence of Cx43 in the cell membrane area. Quantitative analysis of the Cx43-positive membrane intensity in sections from each mouse stain. n = 10. D) Immunostaining for Cx43 (red) for co-culture system of ECs and EGFP-labeled HOBs (green). The nuclei were stained with DAPI (blue). Membrane localization of Cx43 is indicated by yellow arrows. Scale bars, 20 μm. E) Representative endothelial integrity images on the devices treated with GAP19 (5 μm; 30 min) using fluorescence labeled 70 kDa dextran. Scale bars, 100 μm. The below graph demonstrates the endothelial leakiness by diffusive permeability coefficient (Pd) for control and GAP19 treated. The quantitative data are expressed as means ± SD. N = 3, n = 6; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: Primary antibody against rabbit anti-human Cx43 (1:100 dilution in blocking solution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Control, Isolation, Expressing, Quantitative RT-PCR, Western Blot, Membrane, Staining, Immunostaining, Co-Culture Assay, Labeling, Permeability

Journal: Advanced healthcare materials

Article Title: Glucocorticoids Alter Bone Microvascular Barrier via MAPK/Connexin43 Mechanisms.

doi: 10.1002/adhm.202404302

Figure Lengend Snippet: Figure 5. Glucocorticoids regulate microvascular barrier function via MAPK/Cx43. A) Left, Western blot analysis showing GCs-induced activation of Phospho-Cx43Ser282, and Cx43 in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. B) Left, Western blot analysis showing GCs-induced activation of MAPKs (ERK, p38, and JNK) in co-cultures of HOBs and ECs following stimulation with Hyd, Pred, and Psl for 30 min, 60 min, and 120 min. Inhibitor treatment was applied for 30 min. Right, Quantitative analysis of the Western blot results. N = 3. C) Representative images showing endothelial integrity in control and inhibitor-treated group exposed to GCs on the devices for 30 min, using fluorescence-labeled 70 kDa Texas Red dextran. Scale bar, 100 μm. Right panel, Graph demonstrates the endothelial leakiness measured by the diffusive permeability coefficient (Pd) for control and inhibitor- treated groups with GCs. The quantitative data are expressed as means ± SD. N = 3, n = 3; significant differences: *, p-value < 0.05, **, p-value < 0.01, ***, p-value < 0.001.

Article Snippet: Primary antibody against rabbit anti-human Cx43 (1:100 dilution in blocking solution; Cell Signaling Technology) was incubated overnight at 4 °C.

Techniques: Western Blot, Activation Assay, Control, Labeling, Permeability

Journal: bioRxiv

Article Title: Electroanatomical Adaptations in the Guinea Pig Heart from Neonatal to Adulthood

doi: 10.1101/2024.01.26.577234

Figure Lengend Snippet: A) Illustrative examples of ventricular AP activation maps (time of dV/dt max ) in a neonate, younger adult, and older adult guinea pig heart. Electrical pacing (160 ms PCL) was applied near the geometric center, revealing a distinct activation pattern in neonatal hearts. B) Illustrative examples of epicardial CV measured from the earliest to latest activation site, as indicated by the single CV vector (black arrow). Electrical pacing (160 ms PCL) was applied near the apex. C) Epicardial CV restitution curves during S1-S1 pacing (220 to 120 ms PCL), featuring neonates (red, n=7), younger adults (blue, n=11), and older adults (green, n=21). D) Illustration of connexin-43 (red) localization in ventricular myocardium from neonatal and adult guinea pig; nuclei stained with DAPI (blue). 50 μm scale. Replicate values reported as mean ± SEM. Comparisons by two-way ANOVA with multiple comparisons; *p<0.05 compared to neonate (red), young adult (blue), or old adult (green) .

Article Snippet: Tissue slices were blocked with 3% bovine serum albumin, incubated with anti-connexin 43 rabbit primary antibody (1:200; C6219 Sigma Aldrich) overnight at 4°C, and then incubated with a goat anti-rabbit secondary antibody (1:500; Alexa Fluor 647) for 1 hour.

Techniques: Activation Assay, Plasmid Preparation, Staining

Journal: Biochemical and biophysical research communications

Article Title: Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

doi: 10.1016/j.bbrc.2022.11.071

Figure Lengend Snippet: Fig. 1. Nerve injury induced Cx43 and GFAP upregulation in spinal cord. (A) Paw withdrawal thresholds (PWT) was measured with von Frey filaments on sham and SNI mice at baseline and indicated days after SNI operation, n ¼ 6 mice/group. The area under the PWT curve during D1 to D14 of sham and SNI mice were pooled for comparison. (B) Representative blots and quantification of Cx43 protein levels in the lumbar spinal cords of sham and SNI mice at D14, n ¼ 3 mice/group. *P < 0.05, ****P < 0.0001, SNI versus sham. (C) Representative immunofluorescent images of GFAP and Cx43 in the lumbar spinal cords of sham and SNI mice at D14 (bar ¼ 500 mm). Higher amplification GFAP fluorescent images were 3D reconstructed and displayed to show the GFAP immunopositive filaments (Right panels, bar ¼ 50 mm), hypertrophic GFAP filaments were abundant on the surface layer and in the layers II and III. dl, dorsolateral fasciculus.

Article Snippet: Processed samples were separated by SDS-PAGE (10%) electrophorese and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% nonfat milk at room temperature for 1 h, then incubated overnight with primary antibodies at 4 C, Primary antibodies used included rabbit anti-Cx43 primary antibody (3512S, 1:1000, Cell Signaling Technology), and rabbit anti-GAPDH primary antibody (5174S, 1:2000, Cell Signaling Technology).

Techniques: Comparison

Journal: Biochemical and biophysical research communications

Article Title: Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

doi: 10.1016/j.bbrc.2022.11.071

Figure Lengend Snippet: Fig. 2. Cx43 deficiency alleviates mechanical allodynia induced by SNI. (A) Basal mechanical sensitivity, showed as PWT and percentage of positive reaction, was tested in both WT and Cx43/ mice. (B) PWT was measured in Cx43/ mice and C57BL/6 WT at basal level before SNI, D3, D7 and D14. The total area under the PWT curve were pooled from D3 to D14, n ¼ 8. ****P < 0.0001, SNI versus sham. #P < 0.05,##P < 0.01,####P < 0.0001, Cx43/ SNI versus WT SNI. (C) Representative images of GFAP and Cx43 immunoreactivity, and the 3D reconstruction of GFAP immunoreactivity of the lumbar spinal cords of Cx43/ mice at D14 after sham or SNI treatment. Left and middle panels (bar ¼ 500 mm), right panels (bar ¼ 50 mm).

Article Snippet: Processed samples were separated by SDS-PAGE (10%) electrophorese and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% nonfat milk at room temperature for 1 h, then incubated overnight with primary antibodies at 4 C, Primary antibodies used included rabbit anti-Cx43 primary antibody (3512S, 1:1000, Cell Signaling Technology), and rabbit anti-GAPDH primary antibody (5174S, 1:2000, Cell Signaling Technology).

Techniques:

Journal: Biochemical and biophysical research communications

Article Title: Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

doi: 10.1016/j.bbrc.2022.11.071

Figure Lengend Snippet: Fig. 3. Unilateral knockout of spinal cord Cx43 ameliorated SNI-induced mechanical allodynia. (A) Schematic diagram of experimental design. The PWT was measured before (baseline) AAV2/9-GFAP-cre-GFP virus injection and at D3, D7, D14 after SNI operation. (B) Setup for SDH microinjection without laminectomy. (C) Fluorescent of GFP expressed by AAV-cre-GFP viruses, and Cx43 and GFAP expression at 35d after AAV virus injection. Unilateral GFP fluorescent colocalized with Cx43 reduction. bar ¼ 500 mm. (D) PWT was measured in Cx43 fl/flmice with or without virus injection before and after SNI. SNI in Cx43 fl/flmice with SDH injection of AAV-cre-GFP displayed higher PWT, and significantly increased total area under the curve. ****P < 0.0001, n ¼ 8.

Article Snippet: Processed samples were separated by SDS-PAGE (10%) electrophorese and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% nonfat milk at room temperature for 1 h, then incubated overnight with primary antibodies at 4 C, Primary antibodies used included rabbit anti-Cx43 primary antibody (3512S, 1:1000, Cell Signaling Technology), and rabbit anti-GAPDH primary antibody (5174S, 1:2000, Cell Signaling Technology).

Techniques: Knock-Out, Virus, Injection, Microinjection, Expressing

Journal: Biochemical and biophysical research communications

Article Title: Astrocytic connexin 43 deletion ameliorates SNI-induced neuropathic pain by reducing microglia activation.

doi: 10.1016/j.bbrc.2022.11.071

Figure Lengend Snippet: Fig. 4. SNI-induced c-Fos expression and microglia activation in spinal cord is reduced by Cx43 knockout. (A/B) c-Fos-positive cells in the SDH of WT and Cx43/ mice were both significantly increased by SNI, but at lower degree in Cx43/ mice. (C) Representative images of Cx3cr1GFP expressing microglia in the spinal cord at D3 after SNI, SNI significantly alter the microglial cell density and morphology in the ipsilateral SDH over the contralateral SDH. (D) SNI increased the number of ipsilateral microglia in both WT and Cx43/

Article Snippet: Processed samples were separated by SDS-PAGE (10%) electrophorese and transferred onto polyvinylidene fluoride (PVDF) membranes, which were blocked in 5% nonfat milk at room temperature for 1 h, then incubated overnight with primary antibodies at 4 C, Primary antibodies used included rabbit anti-Cx43 primary antibody (3512S, 1:1000, Cell Signaling Technology), and rabbit anti-GAPDH primary antibody (5174S, 1:2000, Cell Signaling Technology).

Techniques: Expressing, Activation Assay, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart

doi: 10.3390/ijms232113073

Figure Lengend Snippet: Apelin-13 treatment increases Cx-43 protein level in NMCs. ( a ) Immunofluorescence evaluation of APJ receptor and TnC expression in NMCs and H9C2 cells. Apelin-13 receptor was stained with anti-APJ antibody (green), and TnC was stained with anti-TnC antibody (red). Cell nuclei were visualised using Hoechst (blue). Images were acquired by confocal laser microscopy at 63×, scale bar 20 μm. ( b ) APJ receptor and TnC expression was also assessed by Western blotting in both NMCs and H9C2 cells; β-actin served as an internal. ( c ) Western blot analysis of Cx43 protein expression in NMCs after cell treatment with different doses of Apelin-13 (10 nM, 100 nM, and 1 μM) for 48 h. Quantitative evaluation of band intensity was expressed as fold change of Apelin-13-treated NMCs vs. untreated cells (Ctr). Vinculin served as internal control (mean ± SEM; *** p ≤ 0.001; n = 3). ( d ) Western blot analysis of Cx43 protein level after NMC treatment with 100 nM Apelin-13 for different timing (6 h, 16 h, 24 h, 48 h, and 72 h). Quantitative evaluation of band intensity was expressed as fold change of Apelin-13-treated NMCs vs. Ctr cells. Vinculin served as internal control (mean ± SEM; *** p ≤ 0.001; n = 3). ( e ) Immunofluorescence analysis of Cx43 protein expression in Apelin-13-treated NMCs (100 nM, 48 h). Cx43 was stained with anti-Cx43 antibody (red), and cell nuclei were visualised using Hoechst staining (blue). Quantitative evaluation of fluorescent dots/cell intensity was expressed as fold change of Apelin-13-treated NMCs vs. Ctr cells (mean ± SEM; * p ≤ 0.05; n = 3). Images were acquired by confocal laser microscopy at 63×, scale bar 20 μm.

Article Snippet: Cells were seeded on coverslips and fixed with 4% PFA, permeabilised with 0.1% Triton X-100, when necessary, and blocked with 5% ( w/v ) BSA and 2.5% ( v/v ) normal goat serum (NGS) for 30 min. NMCs were then stained overnight at 4 °C with the anti-Apelin receptor (APJ) rabbit primary antibody (1:50, by Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-connexin-43 rabbit primary antibody (1:400, purchased from Sigma-Aldrich), anti-LC3B rabbit primary antibody (1:100, from Cell Signaling Technology, Danvers, MA, USA), and anti-TnC mouse primary antibody (1:50, purchased from Santa Cruz Biotechnology) in 1× PBS.

Techniques: Immunofluorescence, Expressing, Staining, Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart

doi: 10.3390/ijms232113073

Figure Lengend Snippet: Apelin-13 increases Cx43 protein levels through autophagy inhibition. ( a ) Quantitative real-time PCR of GJA1 gene in NMCs untreated or treated with Apelin-13 ( n = 3). ( b ) Qualitative immunofluorescence evaluation of LC3 expression in Apelin-13- and 3-MA-treated NMCs vs. Ctr cells. LC3 was stained using anti-LC3 antibody (green), and cell nuclei were visualised using Hoechst (blue). Images were acquired by confocal laser microscopy at 63×, scale bar 20 μm. ( c ) Western blot analysis of p62, Cx43, and LC3 protein expression in NMCs after Apelin-13 treatment (100 nM, 48 h). Quantitative evaluation of band intensity was expressed as fold change of Apelin-13-treated NMCs vs. Ctr cells. Vinculin served as internal control (mean ± SEM; * p ≤ 0.05; *** p ≤ 0.001; n = 4). ( d ) Immunofluorescence analysis of Cx43 protein expression in 3-MA-treated NMCs (5 mM, 16 h). Cx43 was stained with anti-Cx43 antibody (red), and cell nuclei were visualised using Hoechst (blue). Images were acquired by confocal laser microscopy at 63×, scale bar 20 μm. Quantitative evaluation of dots/cell intensity was expressed as fold change of 3-MA-treated NMCs vs. Ctr cells (mean ± SEM; * p ≤ 0.05; n = 3). ( e ) Western blot analysis of p62, Cx43, and LC3 protein expression in NMCs after 3-MA treatment. Quantitative evaluation of band intensity was expressed as fold change of 3-MA-treated NMCs vs. Ctr cells. Vinculin served as internal control (mean ± SEM; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; n = 3).

Article Snippet: Cells were seeded on coverslips and fixed with 4% PFA, permeabilised with 0.1% Triton X-100, when necessary, and blocked with 5% ( w/v ) BSA and 2.5% ( v/v ) normal goat serum (NGS) for 30 min. NMCs were then stained overnight at 4 °C with the anti-Apelin receptor (APJ) rabbit primary antibody (1:50, by Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-connexin-43 rabbit primary antibody (1:400, purchased from Sigma-Aldrich), anti-LC3B rabbit primary antibody (1:100, from Cell Signaling Technology, Danvers, MA, USA), and anti-TnC mouse primary antibody (1:50, purchased from Santa Cruz Biotechnology) in 1× PBS.

Techniques: Inhibition, Real-time Polymerase Chain Reaction, Immunofluorescence, Expressing, Staining, Microscopy, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Apelin-13 Increases Functional Connexin-43 through Autophagy Inhibition via AKT/mTOR Pathway in the Non-Myocytic Cell Population of the Heart

doi: 10.3390/ijms232113073

Figure Lengend Snippet: Apelin-13 increases Cx43 via Akt/mTOR-dependent autophagic pathway. ( a ) Western blot analysis of pathway activated in NMCs by 100 nM Apelin-13 treatment for 30 min and 2 h. Central graph shows quantification of p-Akt/Akt protein expression in Apelin-13-treated NMCs. Vinculin served as internal control (mean ± SEM; * p ≤ 0.05; n = 6). Graph on the right shows quantification of p-mTOR/mTOR protein expression in Apelin-13-treated NMCs (mean ± SEM; * p ≤ 0.05; n = 7). Quantitative evaluation of band intensity was expressed as fold change of Apelin-13-treated NMCs vs. Ctr cells. ( b ) Western blot evaluation of effect of 100 nM Apelin-13 for 30 min, 2 h, and 4 h alone or in combination with 10 μM LY294002 inhibitor. Vinculin served as internal control. ( c ) Western blot analysis of Cx43 protein expression in Apelin-13-treated (100 nM, 48 h) or untreated NMCs with or without LY294002 inhibitor. Quantitative evaluation of band intensity was expressed as fold change of Cx43 protein expression in treated vs. untreated cells. Vinculin served as internal control (mean ± SEM; * p ≤ 0.05; n = 4). ( d ) Western blot analysis of Cx43 protein expression in NMCs either in the presence or absence of p-mTOR inhibitor rapamycin. Cells were treated with inhibitors alone or in combination with 100 nM Apelin-13 for 48 h. Quantitative evaluation of band intensity was expressed as fold change of Cx43 protein expression in treated vs. control cells. Vinculin served as internal control (mean ± SEM; * p ≤ 0.05; n = 3).

Article Snippet: Cells were seeded on coverslips and fixed with 4% PFA, permeabilised with 0.1% Triton X-100, when necessary, and blocked with 5% ( w/v ) BSA and 2.5% ( v/v ) normal goat serum (NGS) for 30 min. NMCs were then stained overnight at 4 °C with the anti-Apelin receptor (APJ) rabbit primary antibody (1:50, by Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-connexin-43 rabbit primary antibody (1:400, purchased from Sigma-Aldrich), anti-LC3B rabbit primary antibody (1:100, from Cell Signaling Technology, Danvers, MA, USA), and anti-TnC mouse primary antibody (1:50, purchased from Santa Cruz Biotechnology) in 1× PBS.

Techniques: Western Blot, Expressing