13-8100

antibody from Invitrogen Antibodies

Targeting: GJB2 CX26, DFNA3, DFNB1, NSRD1

Provider product page for 13-8100

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Antibody data

Antibody Data
Antigen structure
References [74]
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Validations
Western blot [1]
Other assay [38]

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Product number: 13-8100 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 26 Monoclonal Antibody (CX-12H10)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: 13-8100 reacts strongly with Connexin-26, which has a predicted molecular weight of 26.5 kDa. Reactivity of this antibody on western blots has been confirmed by using extracts from mouse liver, mouse brain, and rat brain and it appears to exhibit minimal cross-reaction with Cx30 by western blotting. The degree of cross-reaction with Cx30 by IHC is uncertain, but may be influenced by fixation conditions.
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: CX-12H10
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

GJB2 protein structure - 13-8100 shown in red.

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Experimental details: Figure 1 Immunohistochemical staing for Cx26 in colorectal cancer. Cytoplasmic Cx26 expression was found (x200) .

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Experimental details: Figure 4. Quantitative immunoblot analyses of connexin expression in the adult cochlea. Protein levels were normalized to their corresponding actin bands for quantification. Asterisk in ( A ) indicates a significant reduction in Cx30 in Cx30 T5M/T5M mice compared with Cx30 protein level in wild-type mice. Asterisks in ( B ) indicate a significant reduction in Cx26 in Cx30 T5M/T5M mice compared with Cx26 protein level in wild-type mice.

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Experimental details: Figure 1 Tan IIA treatment of B16 cells results in the upregulation of Cx26 and Cx43 proteins. B16 cells were treated with Tan IIA (0, 5, 10 or 20 uM) for 72 h. (A) Immunoblotting was performed using antibody against Cx26, Cx30 and Cx43. Actin was also tested as a loading control. (B) Relative quantification of the immunoblotting results as calculated by gray scanning (*p

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Experimental details: Figure 5 Mammary tumors from Cx26 knockout mice express similar epithelial protein markers as control mice A. Representative images of paraffin-embedded primary mammary tumor sections immunolabelled with luminal and myoepithelial markers that included; Cx26 (i, ii, iii, red), Cx43 (iv, v, vi, red), keratin 14 (vi, viii green), keratin 8 (vii, green), keratin 10 (ix, red), alpha-smooth muscle actin (x, red), E-cadherin (xi, green) and beta-catenin (xii, red). Hoechst denotes nuclei. Scale bars=50 mum. B. Table indicates relative number of cells that are positive for the luminal and myoepithelial markers based on immunofluorescent labelling. +++50-100%, ++11-49%, +1-10%, -0%.

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Experimental details: Figure 6 DMBA-treated Cx26 knockout mice exhibit similar incidence of metastases to the lungs A. , B. Hematoxylin and eosin stained lung sections were evaluated for evidence of lung tumors revealing a similar proportion of mice that developed lung tumors in Cre+ and Cre- mice. C. Evaluation of average lung tumor area per mouse between Cre+ and Cre- mice revealed the likelihood of Cre- mice having larger lung tumor areas compared to Cre+ mice. D. Representative images of paraffin-embedded lung sections immunolabelled for Ki67 (Red), Cx26 (Red) and Cx43 (Red) revealed a similar percentage of lung tissue expressing high levels of Ki67 positivity between Cre- and Cre+ mice and tumors mostly negative, but not always (insert), for Cx26 and Cx43 expression. Hoechst denotes Nuclei. Scale bars = 50 mum. E. Quantification of Ki67, Cx26 and Cx43 immunofluorescent analysis.

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Experimental details: Figure 3 Macular structure in p63 defective mice and vestibular function in EEC patient ( A ) Representative picture of a macula of the vestibule from mice normal controls stained with MyoVIIa and Connexin 26 antibody. Dapi has been used for nuclei staining. Bars =20 mum. ( B ) Macular neuroepithelium from p63-/- mice. ( C ) Results of vHIT testing of the lateral semicircular canal (LSC) for the patient described in the article; the regression analysis of the vestibulo-ocular reflex (VOR) gain (eye angular velocity/head angular velocity) shows values of 0.48 and 0.45 for the right and left LSC, respectively. This shows VOR hyporeflectivity (average gain between 0.8 and 1.1). ( D ) The vestibular hyporeflectivity for both sides was confirmed by studying VOR gain for all the semicircular canals, with instant gain at 60 milliseconds for the lateral semicircular canals: gain values were all under average, without any significant asymmetry involving one side with respect to the other. Canals positioned on the same plane are connected by lines; RA= right anterior, RP= right posterior, RL=right lateral; LA= left anterior, LP= left posterior, LL=left lateral.

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Experimental details: Figure 1 Expression of Cx26 in HeLa cells. Top row, immunofluorescent staining for Cx26 in HeLa cells stably expressing Cx26. This demonstrates abundant expression of fluorescent puncta representing Cx26 hemichannels and gap junctions. Bottom row, parental HeLa cells do not exhibit any immunofluorescent puncta. In both rows, the corresponding bright-field image is shown on the right. Scale bar 40 mu m.

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Experimental details: The Cx30A88V protein forms gap junction plaques in the plasma membrane of the epidermis. Immunostainings of Cx26 in green (A and B) and Cx30 in red (C and D) in sole skin sections of wildtype and homozygous Cx30A88V mice show that both connexins are expressed in the stratum spinosum and stratum granulosum of the thick epidermis. Both wildtype connexins co-localize (yellow staining in the merged images (E and F)). The mutated Cx30A88V also co-localizes with Cx26 in the plasma membrane (detail view in (F)). Nuclei are in blue. Epidermal layers: St.B: stratum basale, St.S: stratum spinosum, StG: stratum granulosum, St.C: stratum corneum. Scale bars: 20 mum.

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Experimental details: Immunohistochemical expression of alpha-SMA (a, b, c); Cx26 (d, e, f); Cx43 (g, h, i), and Cx30 (j, k, l) in tissue sections from the involutional phase of DD (a, d, g, j); in tissue sections from the residual phase of DD (b, e, h, k), and in tissue sections from controls (patients with carpal tunnel syndrome) (c, f, i, l) (magnification 400x).

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Experimental details: FIGURE 1 Characterization of connexin expression and Cx43 ablation in HEI-OC1 cells. (A) Western blots for Cx43, Cx26, and Cx30 protein in wild type (WT) and three independent clones of CRISPR-Cas9 Cx43-knockout (Cx43-KO) HEI-OC1 cells where adult mouse cochlear lysate was used as a positive control. Molecular weight standards are denoted in kDa. (B) Immunolabeling revealed Cx43 gap junctions only in WT cells denoted by white arrows. Note Cx26 and Cx30 were not detected in WT or Cx43 KO cells (A,B) . Red = Cx43, Cx26, and Cx30, green = phalloidin staining of actin filaments, blue = Hoechst stained nuclei. Bars = 10 mum.

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Experimental details: Figure 5 Representative Examples of Co-localization Imaging for Connexin 26 with a Cell Group of Interest (A) A positive control for successful connexin 26 staining (leptomeninges, red). (B) Co-localization staining of connexin 26 (red; 594) with glial cells (GFAP; green; 488). Images were acquired on a Leica 880 confocal and processed using Zen Black software following the above immunohistochemistry protocol detailed above. Scale bars, 50 mum. Figure reproduced in part using content previously published in ; iScience on a CC-BY 4.0 license.

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Experimental details: 4 Distribution of Cx26 in subependymal and subpial zones. A: Intense Cx26-ir punctation (antibody 71-0500; green) in the subependymal layer (SEL) of the third ventricle (3V). Note the decreasing gradient of intensity from the ependymal borderline. Ependymocytes (Ep) and blood vessels (arrows) appear pink due to immunoreactivity for both S100beta (red) and vimentin (blue). B: Intense immunoreactivity for basic fibroblast growth factor (bFGF) in the nuclei of astrocytes in the SEL of 3V. C: The presence of numerous Cx26-ir puncta (antibody 51-2800) in meningeal projections (M) of the lateral ventricle (LV). Puncta were small in the SEL but large on the periphery of blood vessels (BV). Note the lines of small, Cx26-ir puncta along the arrows. D: Immunoreactivity for Cx26 (antibody 51-2800; green) and Cx43 (antibody 13-8300; red) was detected either as double-labeled puncta (arrows) or as single puncta (arrowheads) in the SEL of 3V. The ependyma was primarily Cx43 immunoreactive. E: Distribution of Cx26 immunoreactivity (antibody 13-8100) in the supraoptic nucleus (SON) and underlying meninges. Note the similar concentration of Cx26-ir puncta in the meninges and ventral glial limitans (vgl). White dashed lines indicate the brain surface. Cx26 immunoreactivity was intense in proximity to BV. OT, optic tract; Amyg, amygdala. Scale bars = 20 mum in A; 25 mum in B-D; 50 mum in E.

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Experimental details: 6 Association of Cx26 and bFGF in meningeal, subpial, and perivascular layers. All images show Cx26 immunoreactivity (antibody 13-8100; red) and bFGF immunoreactivity (green). A: Section of the periamygdaloid cortex showing an association of Cx26 immunoreactivity and bFGF immunoreactivity in the VGL (arrows), pia (P), and arachnoid (A). White lines indicate the brain surface. Note a Cx26-ir punctum in proximity to the nucleus of a pial fibroblast (arrowhead). BV, blood vessel. B: Magnification of the previous field. Note the continuum of Cx26-ir puncta between the pia and the vgl. An arrowhead indicates the brain surface. C: High concentrations of immunoreactivity to bFGF and Cx26 in the vgl of the ventromedial hypothalamus (VMH) and in the periphery of BV. Note that Cx26-ir puncta were associated (arrowheads) or were not associated (arrow) with nuclear bFGF immunoreactivity. D: Cx26-ir puncta and bFGF immunoreactivity in a meningeal projection (M) between the thalamus (Th) and the fimbria hippocampus (FH). Note the intense nuclear bFGF immunoreactivity in astrocytes that face the meningeal projection (arrow). bFGF immunoreactivity was less intense in astrocytes that were distant from the border zone (large arrowhead) and in fibroblasts (arrowhead). Scale bars = 20 mum in A; 10 mum in B; 25 mum in C,D.

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Experimental details: Figure 3 Dye Loading and Cx26 Expression in Substantia Nigra (A) Carboxyfluorescein (CBF) dye loading following hypercapnia in P7-10 slices (cell bodies are clearly labeled). No dye loading occurred if CO 2 was not changed or in P17-21 slices; scale bar, 50 muM. (B) Immunofluorescent staining of P7-10 SN for Cx26 (red, arrows) in TH + neurons (green). (C) No co-localization of Cx26 (red) in TH + neurons (green) in the SN at P17-21; scale bars, 30 muM. Staining was deemed successful due to the positive leptomeninges staining from corresponding sections of the same brain. (D) Scale bar, 50 muM. (E) Cx26 (red, arrows) co-localized with GFAP (green, glial cell marker) in P17-21 SN; scale bar, 50 muM.

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Experimental details: Figure 4 GABAergic Neurons in the VTA Are Sensitive to CO 2 (A) CBF dye loading of VTA neurons in response to hypercapnia occurs at both P7-10 and P17-21 but does not occur without the increase in CO 2 (hypercapnia), scale bar, 50 muM. (B) Characteristics of CO 2 -sensitive VTA neurons: firing pattern, hyperpolarization to the opioid receptor agonist [Met5]Enkephalin (10 muM) but not to dopamine (30 muM). (C) Time course of changes in voltage response (CO 2 increased from 35 to 55 mm Hg, each point is a mean of six current steps, error bars are SEM). (D) Voltage responses to step currents at indicated time points in (C). (E) Quantification of changes in voltage response to increased CO 2 . (F) Time course of changes in voltage response (CO 2 decreased from 35 to 20 mm Hg, each point is a mean of six current steps, error bars are SEM). (G) Voltage responses to step and fluctuating current inputs (as in ; see Methods ) at indicated time points in (F) demonstrating increased input resistance and firing rate. (H) Quantification of changes in voltage response to decreased CO 2. (I and J) Representative single optical planes immunohistochemistry images. (I) Immunofluorescent staining of P17-21 VTA for Cx26 (red), which is not expressed by TH + neurons (green, no co-localization); scale bar, 50 muM. (J) Co-localization of Cx26 (red) with the soma of three individual GAD + neurons (green) in the VTA (scale bar, 20 muM).

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Experimental details: Figure 5 3D Organotypic epidermal model. Keratinocytes grown in transwells on either a normal (column NK/NF) or on a psoriatic (column NK/PF) fibroblast feeder layer were fixed and stained after 12 days at the ALI with relevant antibodies followed by 3D z-stack reconstruction (Z: NK/NF). IHC analysis determined expression of ( a ) CX26, ( b ) CX43, ( c ) Ki67, ( d ) E-cadherin and ( e ) CK16 protein expression. ( a - e ) bar = 100 mum. The mean fluorescent intensity of multiple regions from different cultures was extracted (right hand panel). Statistical analysis was performed by Student t -test N = 3. * p < 0.05; ** p < 0.001.

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Experimental details: Immuncytochemical characterization of the purified fibroblasts (P3 after 72 h cultivation). (A) Anti-Vimentin, clone V9 (Cy5, red, 3 s). (B) Anti-S100 (Cy3, yellow, 400 ms). (C) Anti-Cx26 (Cy5, red, 5 s). (D) Anti-carbonic-anhydrase II (Cy3, yellow, 100 ms). All preparations were additionally stained for DAPI (blue, 1 s).

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Experimental details: 2 FIGURE Localization of CX26 mutations and the impact on CX43 and the microtubule network. HaCaT cells (A) and HeLa43 (B) were transfected with CX26WT-GFP (i), CX26F142L-GFP (ii), CX26G12R-GFP (iii) or CX26D66H-GFP (iv) (Cx -green) and co-stained for Cx43 (red). HeLa26 cells (C) were transfected with CX26WT-mCherry (red) (i), CX26F142L-mCherry (red) (ii), CX26G12R-GFP (green) (iii) or CX26D66H-GFP (green) (iv) and co-stained CX26 (stained green in Ci, Cii and red in Ciii and Civ). Areas of co-localization stained yellow. White arrow indicates trapped F142L, and yellow arrow indicates Cx43 Gap Junction staining. HeLa26 cells (D) were transfected with CX26WT-GFP (green) (i), CX26F142L-mCherry (red) (ii), CX26G12R-GFP (green) (iii) or CX26D66H-GFP (green) (iv) and were co-stained for -tubulin (stained red Di, Diii, Div and green in Dii). HeLa26 cells (E) were transfected with CX26WT-GFP(i), CX26F142L-mCherry (ii), CX26G12R-GFP (iii) or CX26D66H-GFP (iv) and treated with paclitaxel throughout the transfection period followed by co-staining with beta-tubulin (stained red Ei, Eiii, Eiv and green in Eii). Nuclei were stained with DAPI (blue). Scale bar = 10 um

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Experimental details: Fig 3 Expression of the gap-junction proteins connexin 26, connexin 32, and connexin 43 in H69 cells after treatment with NDMA and/or ESP, as determined by western blotting. H69 cells were incubated with either PBS (vehicle) or NDMA and/or ESP for 72 h, and the cells were collected for protein extraction. A. The blots of each groups were run under same experimental conditions and the images were cropped from different parts of the same gels. B-D. Quantification of relative Cx43 Cx26, and Cx32 expression in each group. The each proteins level are indicated as normalization of the ratio of Cx43/Calnexin, Cx26/Calnexin, and Cx32/Calnexin, and Cox-2/Calnexin. DATA represent the mean +- SE of five independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control.

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Experimental details: Fig 4 Concentration of intracellular connexin 26, connexin 32, and connexin 43 in human cholangiocytes (H69 cells). After treatment with NDMA and ESP for 72 h, the concentration of intracellular connexin 26, 32, and 43 in H69 cells was measured by laser scanning microscopy (x4,000). A-C: Immunofluorescence confocal microscopy indicated that the intracellular concentrations of Cx26 (green)/DAPI (blue), Cx32 (blue)/ DAPI (red), and Cx43 (green)/ DAPI (red) increased in cells treated with NDMA + ESP. Scale bar = 25 mum. Data represent the mean +- SE of five independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001 versus control.