51-2800

antibody from Invitrogen Antibodies

Targeting: GJB2 CX26, DFNA3, DFNB1, NSRD1

Provider product page for 51-2800

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Antibody data

Antibody Data
Antigen structure
References [64]
Comments [0]
Validations
Western blot [1]
Immunocytochemistry [1]
Immunohistochemistry [2]
Other assay [67]

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Product number: 51-2800 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 26 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 0.25 mg/mL
Storage: -20°C

GJB2 protein structure - 51-2800 shown in red.

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Experimental details: Immunohistochemistry analysis of Connexin 26/GJB2 showing staining in the cytoplasm of paraffin-embedded mouse colon tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Connexin 26/GJB2 polyclonal antibody (Product # 51-2800) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Immunohistochemistry analysis of Connexin 26 showing staining in the membrane of paraffin-embedded mouse liver tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Connexin 26 Polyclonal antibody (Product # 51-2800) diluted in 3% BSA-PBS at a dilution of 1:20 overnight at 4ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

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Experimental details: Figure 5 Recovery of Cx26 expression in Cx26 Sox10Cre organotypic cultures transduced with BAAVCx26CFP. Top, immunoreactivity to Cx26 antibodies in a representative Cx26 loxP/loxP culture. Middle, lack of Cx26 immunoreactivity in a representative Cx26 Sox10Cre culture. Bottom, immunoreactivity to GFP antibodies, which also recognize CFP, in a representative Cx26 Sox10Cre culture transduced with BAAVCx26CFP. In all panels, actin filaments were stained with Texas red conjugated phalloidin (red). Scale bar, 10 um.

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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 8 Effect of hyperglycaemia on staining of immunoreactive Cx proteins in cultured astrocytes Composite z-stacks of confocal images (left column) of immunostained astrocytes showed a low-intensity background and prominent staining of punctate or vesicular immunoreactive material that appeared to be mainly intracellular. This morphological appearance of immunostaining was evident for Cx43 ( A ), Cx30 ( B ) and Cx26 ( C ) protein in astrocytes grown on coverslips for 14 days in a medium containing 5.5 mmol/l glucose; the scale bar is 12.5 mum and applies to all panels. Areas of these immunostained punctate/vesicular objects (right columns) are means (vertical bars represent 1 S.D.) from the following numbers of objects per group: Cx43, 5.5 mmol/l: n = 752 objects in cells on five coverslips; 25 mmol/l: n = 884 objects in cells on five coverslips; Cx30, 5.5 mmol/l: n = 1099 objects, five coverslips; 25 mmol/l: n = 1177 objects, five coverslips; Cx26, 5.5 mmol/l: n = 514 objects, four coverslips; and 25 mmol/l: n = 974 objects, five coverslips.

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Experimental details: Figure 3. Connexin immunoreactity in the adult organ of Corti. Maximal projection rendering of two consecutive midmodiolar confocal optical sections, taken at 1 mum intervals in the indicated cochlear turns of Cx30 T5M/T5M (left) and Cx30 +/+ mice (right) on P30. Cx30 and Cx26 expression was analysed with selective antibodies (green), nuclei were stained with DAPI (blue) and actin filaments with Texas red conjugated phalloidin (red). Scale bar, 50 mum.

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Experimental details: Figure 1 BLG-Cre; Cx26 fl/fl mice exhibit a dramatic reduction in Cx26. (A) Real-time PCR analysis of wild-type mice revealed that Cx26, Cx32 and Cx30 are upregulated at parturition and lactation. (B, C) Real-time PCR and Western blot analysis of mammary glands from control (open columns) and Cre-treated (solid columns) mice revealed a dramatic reduction in Cx26 mRNA and protein levels at partition and lactation. *p

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Experimental details: Fig 7 Lactating Panx1 -/- mice have fewer Cx32 gap junctions in the mammary gland. (A) Immunofluorescent analysis of mammary gland cryosections during early lactation for Cx26, Cx30 and Cx32 (red) and cytokeratin14 (green) revealed no change in Cx26 and Cx30 gap junctions in knockout mice, while fewer Cx32 gap junctions were observed compared to control mice. Hoescht (blue) denotes nuclei. Scale bar = 50 um. (B) Values represent the mean number of connexin plaques (red) relative to the pixel area of the nuclei (blue), multiplied by a factor of 1x10 2 , per 0.3mm 2 +- S.E.M. N = 5.

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Experimental details: Figure 3 Confocal immunofluorescence imaging of cochlear cross-sections from mice injected at P25 with BAAVCre-IRESGFP viral vectors. Color code: Cx26, green; actin filaments, red; nuclei, blue. ( a,b ) Stria vascularis (SV) and spiral ligament (SL). ( c,d ) organ of Corti; HCR, hair cell region. ( e,f ) close-up view of the HCR from c,d, respectively. DCs: Deiters' cells; IHC: inner hair cell; OHCs: outer hair cells; PCs: pillar cells. Scale bars: 50 mum in ( a-d ); 25 mum in ( e,f ).

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Experimental details: Figure 5 Recovery of Cx26 expression in Cx26 Sox10Cre organotypic cultures transduced with BAAVCx26CFP. Top, immunoreactivity to Cx26 antibodies in a representative Cx26 loxP/loxP culture. Middle, lack of Cx26 immunoreactivity in a representative Cx26 Sox10Cre culture. Bottom, immunoreactivity to GFP antibodies, which also recognize CFP, in a representative Cx26 Sox10Cre culture transduced with BAAVCx26CFP. In all panels, actin filaments were stained with Texas red conjugated phalloidin (red). Scale bar, 10 um.

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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: Representative immunohistochemistry of Cx26, Cx30, and Cx43 in sinus mucosa of control and CRS patients. The white dotted line demarcates the epithelium and stroma. Nuclei were stained with DAPI (blue). (A, B) Paired control and CRS patient sections with Cx26 staining (green) around the basal epithelial nuclei. (C, D) Paired control and CRS patient sections with Cx30 staining (green) through the epithelial layers, with some green autofluorescence within the stroma. (E) Minimal Cx43 in the stroma (white circles) of a control patient. (F) CRS patient showed massive elevation of Cx43 in the stroma. CRS = chronic rhinosinusitis; DAPI = 4',6-diamidino-2-phenylindole.

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Experimental details: Figure 2 Rat epidermal keratinocytes (REKs) express endogenous Cx43 and retain the capacity to assemble GFP-tagged Cx26, Cx30 and Cx31 into gap junctions. ( A ) Basal keratinocyte-like REKs express endogenous Cx43, trace amounts of Cx26 (arrows) but no detectable levels of Cx30 or Cx31 protein. REKs engineered to express GFP-tagged connexins were used to demonstrate that the anti-connexin antibodies were efficient at detecting connexins with the exception of the anti-Cx31 antibody. ( B ) REKs engineered to express GFP-tagged Cx43, Cx26, Cx30 and Cx31 all assembled prototypical gap junctions characterized by small punctate structures at sites of cell-cell interface (arrows). In addition, an array of intracellular fluorescent structures could be identified that reflect organelles and vesicles involved in connexin transport. Nuclei were stained with Hoechst dye. Bars = 10 mum.

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Experimental details: Figure 3 Ablation of Cx43 did not act to stimulate connexin reprogramming or differentiation of REKs. ( A ) CRISPR-Cas9 was effectively used to ablate Cx43 from REKs as revealed by immunoblotting wild-type (WT) and three separate clones (C1, C2, C3) for Cx43. Immunoblotting for GAPDH was used as a loading control. ( B ) Immunofluorescent labeling for Cx43, Cx26 and Cx30 revealed that Cx43 was ablated from all three CRISPR-Cas9 treated clones and neither Cx26 nor Cx30 exhibited increased expression to compensate for the loss of Cx43. Immunolabeling for keratin 10 (K10) and keratin 14 (K14) revealed that REKs retained their basal cell characteristics upon Cx43 ablation. Inserts represent transverse or en face sections of mouse skin as positive controls for anti-Cx30 and anti-K10 antibodies while normal rat kidney cells engineered to express Cx26-GFP were used as a positive control for the anti-Cx26 antibody. Nuclei were stained with Hoechst dye. Bars = 10 mum.

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Experimental details: Figure 1 Localization of Cx26 in several newly established HeLa cell lines (confocal micrographs). The panels of Cx26 variants are designated (top down) as KO, WT, W172C, R75Q, and 35delG for cell lines HeLa Cx26-KO, HeLa-Cx26wt, HeLa-p.W172C, HeLa-p.R75Q, and HeLa-c.35delG, respectively. Nuclei were visualized with DAPI (blue), transgenic HeLa cells were visualized with Green Fluorescent Protein (GFP) and Cx26 was immunostained with antibodies against C-terminus of Cx26 (red). Scale bar = 20 um.

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Experimental details: Figure 2 A fraction of HeLa-p.W172C cells with reduced number of protein granules (confocal micrographs). Blue color indicates DAPI nuclei staining, green color corresponds to GFP signal, red color corresponds to Cx26 signal. Scale bar = 10 mum.

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Experimental details: Figure 3 Analysis of the Cx26 protein expression in transgenic HeLa cell lines. The total lysates of cells were subjected to Western blot analysis with antibodies against Cx26 and the loading control, alpha-tubulin. The panels of Cx26 variants are designated as KO, WT, W172C, R75Q, and 35delG for cell lines HeLa Cx26-KO, HeLa-Cx26wt, HeLa-p.W172C, HeLa-p.R75Q, and HeLa-c.35delG, respectively.

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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: Figure 1 Molecular volumes obtained for homomeric Cx26-HA, heteromeric Cx26-HA/Cx30, and Cx30-HA/Cx26 hemichannels from AFM imaging. a , identification of purified hemichannels. Upper , detection of Cx30-HA/Cx26, Cx26-HA/Cx30, and Cx26-HA protein in eluates from HA-agarose columns. Samples were analyzed by SDS-PAGE and immunoblotting, using mono and polyclonal anti-HA and anti-Cx26 primary Ab, respectively, followed by a horseradish peroxidase-conjugated goat anti-mouse and rabbit secondary Ab, respectively. Lower , peptide sequences identified by MS analysis after tryptic digestion from heteromeric Cx30-HA/Cx26 hemichannels, corresponding to sequences found for both Cx30 and Cx26 isoforms. b , low-magnification image ( scale bar , 50 nm). c , high-magnification images of single proteins. Sections through particles are shown as red lines including two points, height and radius at half height ( blue and green arrows , respectively) ( scale bar , 20 nm). d , particle height analysis of the indicated section. e , frequency distribution of molecular volumes. Black curves indicate fitted Gaussian functions.

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Experimental details: Figure 5 Immunohistochemical staining of Cx26 and Na + /K + -ATPase in rats treated with NS, FSK, ATRA, or FSK + ATRA under CDDP exposure. Representative images of Cx26 (brown, DAB) in the OC ( A ), SGNs ( B ), and LW fibrocytes ( C ) obtained from the middle turn of the cochlea (n = 5 per group). ( D ) Representative image of Na + /K + -ATPase in LW fibrocytes. The cells were counterstained with hematoxylin (blue). The inset shows a magnified image of the square box. ( E ) Quantification of the chromogenic intensity in the OC, SGNs, and LW fibrocytes in the middle turn of the cochlea in each group. Scale bar = 20 mum * p < 0.05, ** p < 0.01, *** p < 0.001. NS, normal saline; CDDP, cisplatin; FSK, forskolin; ATRA, all-trans retinoic acid; OC, organ of Corti; SGNs, spiral ganglion neurons; LW, lateral wall; Cx26, connexin26.

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Experimental details: Figure 2 Connexin (Cx) hemichannels (HCs) expressed in melanoma cells mediate the propagation of calcium (Ca 2+ ) waves induced by focal photodynamic therapy (fPDT) in vivo. ( A ) Pooled results of fPDT trials in GCaMP6s-expressing dorsal skinfold chamber (DSC) tumors in the following conditions: Ca 2+ -free extracellular medium (CFEM) supplemented with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA, 5 mM); normal extracellular medium containing 2 mM of Ca 2+ (NEM, control); CFEM supplemented with EGTA (5 mM) plus carbenoxolone (CBX, 100 uM) or flufenamic acid (FFA, 100 uM) or TAT-Gap19 (150 uM) or abEC1.1m (1 muM). The histogram shows the area under GCaMP6s Delta F / F 0 traces ( A , inset) computed between the onset of fPDT ( t = 10 s) and the end of the observation time window ( t = 80 s) for each bystander cell order (abscissa); pooled data [mean +- standard error of the mean (s.e.m.)] for n >= 6 experiments in at least 2 different tumors for each condition. a.u., arbitrary units; *, p < 0.05; ***, p < 0.001, the Kruskal-Wallis test (for post hoc pairwise comparisons, see Table S1 ). Data for EGTA and NEM conditions are also shown in Figure 1 C; ( B - D ) In vivo 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI) uptake experiments: GCaMP6s-expressing DSC melanomas were incubated with DAPI (5 muM) dissolved in: CFEM supplemented with 5 mM of EGTA; NEM; CFEM supplemented with 5 mM of EGTA plus FFA (100 uM) or TAT-Gap19 (150 uM) or abEC1.1m (

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Experimental details: Figure 2 Connexin protein expression in cholestasis. Semi-quantitative immunoblot analysis of Cx26, Cx32 and Cx43 species in livers of cholestatic mice ( A ) and in human hepatoma HepaRG cells cultured in cholestatic conditions ( B ) was performed. For Cx43, both the phosphorylated (P) and non-phosphorylated (NP) variant could be detected. Signals of the three connexins were normalized against total protein loading and expressed as relative alterations compared to sham-operated animals or to control samples, respectively. ( A ) Liver sections were obtained from male mice following bile duct ligation (BDL) for 20 days. Data were processed by a parametric student t-test with Welch's correction or a non-parametric Mann-Whitney test. Data are expressed as means +/- SD with * p

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Experimental details: Figure 5 Connexin protein localization in livers of cholestatic mice. Liver sections were obtained from male mice following bile duct ligation (BDL) for 20 days. Cellular localization of Cx26, Cx32 and Cx43 (green) was revealed by immunohistochemistry analysis with nuclear counterstaining using DAPI (blue). Scale bar, 100 um (Sham n = 3; BDL n = 3) (N = 1).

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Experimental details: Figure 8 Total protein loading of samples used in immunoblot analysis. Semi-quantitative immunoblot analysis of Cx26, Cx32 and Cx43 in liver of cholestatic mice ( A ) and in human hepatoma HepaRG cells cultured in cholestatic conditions ( B ) was performed. For Cx43, both the phosphorylated (P) and non-phosphorylated (NP) variant could be detected. Signals of the three connexins were normalized against total protein loading, which are shown for two representative samples. ( A ) Liver sections were obtained from male mice following bile duct ligation (BDL) for 20 days (Sham n = 12; BDL n = 18) (N = 1). ( B ) Human hepatoma HepaRG cells were exposed to cholestatic drugs either in the absence or presence of a 50x concentrated mixture of bile acids (BA) for 72 h and compared to untreated human hepatoma HepaRG cells, indicated in the figure as control. The different experimental conditions are presented in the figure as: 1 = control ( n = 3); 2 = control BA ( n = 3); 3 = atazanavir (ATV) ( n = 3); 4 = ATV BA ( n = 3); 5 = cyclosporine A (CsA) ( n = 3); 6 = CsA BA ( n = 3); 7 = nefazodone (NEF) ( n = 3); 8 = NEF BA) ( n = 3) (N = 1).

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Experimental details: Figure 1 Cx26, Cx32, and Cx43 protein expression in human hepatoma HepaRG cells compared to primary human hepatocytes (PHH). Proteins were extracted from human hepatoma HepaRG cells ( n = 3 and N = 3) and PHH ( n = 1 and N = 3) and immunoblotting was performed. Densiometric quantitative data were obtained via Image Lab 6.0.1 software (Bio-Rad, Hercules, CA, USA). Data were normalized to the total protein loading ( Figure S1 ) and expressed as a ratio to PHH (if the target was expressed in PHH). Significant differences compared to PHH were calculated with a parametric one-way analysis of variance (ANOVA) followed by a Dunnett's post-hoc test to correct for multiple comparisons. Data are expressed as mean +- standard deviation with * p

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Experimental details: Cx26 protein expression in human hepatoma HepaRG cells exposed to GTX, NGTX, and NC chemicals. Human hepatoma HepaRG cells ( n = 3 and N = 1) were exposed to (non)-carcinogenic chemicals for 72 h. Densiometric quantitative data were obtained via Image Lab 6.0.1 software (Bio-Rad, Hercules, CA, USA). Data were normalized to the total protein loading ( Figure S2 ) according to Bio-Rad's instructions [] and expressed as a ratio to their respective solvent control (DMSO CTL or PBS CTL). Significant difference compared to the solvent control (DMSO CTL or PBS CTL) was calculated with a parametric one-way ANOVA followed by a Dunnett's post-hoc test to correct for multiple comparisons. Data are expressed as mean +- standard deviation with * p

Supportive validation

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Experimental details: Cx26 protein localization in human hepatoma HepaRG cells exposed to GTX, NGTX, and NC chemicals. Human hepatoma HepaRG cells ( n = 1 and N = 1; at least three images per well) were exposed to GTX, NGTX, and NC chemicals for 72 h. The area and intensity of the fluorescent signal was quantified via ImageJ software (version 1.52p, Bethesda, MD, USA) and normalized to the fluorescent signal of the respective solvent control (DMSO CTL or PBS CTL). Significant difference compared to the solvent control was calculated with a parametric one-way ANOVA followed by a Dunnett's post-hoc test to correct for multiple comparisons. Data are expressed as mean +- standard deviation with * p

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Experimental details: Figure 3 Cx26 ( A ), Cx32 ( B ) and Cx43 ( C ) protein expression in liver cancer cell lines and primary human hepatocytes (PHH). Cancer cell lines ( n = 1, N = 3) were grown to 100% confluence, while PHH were used in suspension for protein extraction. Immunoblotting and visualization were done with the Pierce(tm) ECL Western Blotting Substrate kit (Thermo Fisher Scientific, Waltham, MA, USA) on a ChemiDoc TM MP imaging system. All signals were divided by their respective total protein loading signal and normalized by the sum of all data points in a replicate []. Unlike Cx43, which was not expressed by PHH, Cx26 and Cx32 are expressed relative to their expression in PHH. Data are expressed as mean +- standard deviation with * p

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Experimental details: Figure 2 Identification of novel Cx32 interactors by IP-HTMS ofCx32-enrichedliver membrane fractions and validation by co-IP of tissue lysates.(A) Silver stain of proteins found in sucrose gradient fractions 4-6(Figure 1 B) following IP with monoclonal anti-Cx32(Table 1 ). Negative controls included IP reactionsfrom KO fractions 4-6 (Supplemental Figure 1, Supporting Information ) and a no lysate (NL) mock IP reaction.Black boxes indicate gel regions excised for tryptic digest. Mobilityof precipitating IgG heavy and light chains are indicated by arrowheads.(A') Western blot of Cx32-enriched fractions 4-6 (F4-6,5 mug) and 1 muL (10%) of the corresponding IP productsfrom these same fractions analyzed in (A). Analysis confirmed presenceof bait (Cx32) in WT samples and lack of cross-reactivity in KO samples.Cx32-M indicates use of monoclonal anti-Cx32; Cx32-P indicates useof polyclonal anti-Cx32 antibody (Table 1 ).(B) IP-HTMS identified one known (Cx26) and 17 novel Cx32 interactingpartners in replicate screens of 1 mg and 5 mg input protein. Interactionnetwork generated by Osprey version 1.2.0 of proteins present in WTbut not KO IP-HTMS. Proteins identified in both the 1 mg and 5 mgscreens are represented with larger icons and thicker interactionlines than proteins identified only in the 5 mg screen. (C) Reciprocalco-IPs of liver lysates for Cx32 and Cx26. (D) Reciprocal co-IPs forCx32 and SFXN-1. Specificity was also verified in independent IPsusing an isotype IgG control

Supportive validation

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Experimental details: Fig. 1 Distribution patterns of Cx26 in different SCs of the mouse cochlea at different postnatal time points. a - d Postnatal Cx26 immunolabeling (red) in cochlear radial sections. The yellow box in a indicates the location of the organ of Corti. e - h Postnatal Cx26 immunolabeling (red) in radial sections of the organ of Corti. Asterisks indicate the different rows of DCs. D1:DC1, the first row of DCs; O1:OHC1, the first row of OHCs. White arrows and arrowheads indicate different SCs that show Cx26 expression. i - k Postnatal Cx26 immunolabeling (red) in flattened preparations. l - n Postnatal Cx26 (red) and Sox2 (white) immunolabeling in corresponding flattened preparations. Images of the radial section (left) are given to show the levels of the images. The white arrowheads indicate Cx26 expression on the radial sides of DCs. The white arrows indicate longitudinal Cx26 expression in DCs. o Cx26 immunolabeling in ISCs at P3. p Cx26 immunolabeling in different rows of DCs at P3. q Cx26 immunolabeling in CCs at P3. Images of the radial section are shown on top of o , p , and q , and the yellow boxes in the images are the areas of magnification in o , p , and q . GER great epithelial ridge, ISC inner sulcus cell, PC pillar cell, IHC inner hair cell, OHC outer hair cell, CC Claudius cell. The scales in panels a , e , i , and o represent 100, 40, 30, and 10 um, respectively

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Experimental details: Fig. 2 Cx26 expression patterns in different experimental groups. a , b The tdTomato staining (red) shows that Cre was activated in cells in the cochlea ( a ) and the organ of Corti (OC) ( b ). c Representative western blot results show Cx26 expression in the control groups, the randomly Cx26- group (R group, R), and the specific longitudinally Cx26- group (L group, L) at P7. d - f Cx26 immunolabeling in the apical, middle, and basal turns of the control group, respectively. g - i Cx26 immunolabeling in the apical, middle, and basal turns of the R group, respectively. j - l Cx26 immunolabeling in the apical, middle, and basal turns of the L group, respectively. Asterisks indicate the Cx26- DCs. The white arrowheads indicate the region of Cx26- OPCs, whereas the white arrows indicate the region of Cx26- IPCs. m Relative expression of cochlear Cx26 at P7 ( n = 4 in each group) in the control and experimental groups. n Cx26- cell counts at P7 in different turns of the cochlea from different experimental groups ( n = 3 in each group). o Cx26- DC counts at P7 ( n = 3 in each group). *Significant difference between two groups ( P < 0.05). SV striae vascularis, SGN spiral ganglion neuron. The scales in panels a , b , and d represent 100, 30, and 30 um, respectively

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Fig. 5 Cx26 expression patterns and OHC loss patterns in different longitudinally Cx26- groups. a - c Cx26 immunolabeling (red) in the apical, middle, and basal turns of the longitudinally Cx26- group (1/3), respectively. d - f Cx26 immunolabeling in the apical, middle, and basal turns of the longitudinally Cx26- group (1/6), respectively. Asterisks indicate Cx26- DCs. g Cx26- cell counts at P7 in different turns of the organ of Corti ( n = 3 in each group). h Cx26- DC3 counts at P7 ( n = 3 in each group). * and # indicate significant differences between two groups ( P < 0.05). i - k Representative images of HCs in the apical ( i ), middle ( j ), and basal turns ( k ) from the longitudinally Cx26- group (1/3). l - n Representative images of HCs in the different turns from the longitudinally Cx26- group (1/6). o Quantifications of OHC3 loss at specific cochlear locations in different groups at P18. Asterisk indicates a significant difference compared with the specific longitudinally Cx26- group. The hash symbol indicates a significant difference compared with the longitudinally Cx26- group (1/3). p - s Representative images of the middle-basal parts of flattened cochlear preparations in different groups. White arrowheads indicated the regions of OHC3 loss. The scales in panels a , i , and p represent 30, 40, and 200 um, respectively