71-0700

antibody from Invitrogen Antibodies

Targeting: GJA1 CX43, GJAL, ODD, ODDD, ODOD, SDTY3

Provider product page for 71-0700

Western blot

ELISA

Immunohistochemistry

Other assay

Antibody data

Antibody Data
Antigen structure
References [237]
Comments [0]
Validations
Western blot [3]
Immunohistochemistry [4]
Other assay [29]

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Product number: 71-0700 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 43 Polyclonal Antibody
Antibody type: Polyclonal
Antigen: Synthetic peptide
Description: This antibody reacts with connexin 43. On western blots, this antibody detects a 43 kDa protein with mouse brain, rat brain, and human smooth muscle cell extracts. Reactivity with other connexin types has not been observed on western blots (note: samples were not boiled prior to running SDS-PAGE). Positive immunohistostaining is seen with mouse heart and thalamus tissue. Paraffin embedded sections require a heat-induced epitope retrieval (HIER) step. Reactivity of this antibody with connexin 43 is independent of phosphorylation status.
Reactivity: Human, Mouse, Rat
Host: Rabbit
Isotype: IgG
Vial size: 50 µg
Concentration: 0.25 mg/mL
Storage: -20°C

GJA1 protein structure - 71-0700 shown in red.

Supportive validation

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Experimental details: Western blot analysis of Connexin43 (Cx43) was performed by loading 30 µg of mouse heart (not boiled) and 7 µL of molecular weight protein ladder per well onto a 10% Tris-Hcl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked in blocking buffer composed of 20 mM Tris-HCl pH 8, 150 mM NaCl and 0.2% Tween-20 (TBST) supplemented with 5% skim milk powder (Carnation) for 1 hr at room temperature. Cx43 was detected at ~43 kDa using an anti-Cx43 rabbit polyclonal antibody (Product # 71-0700) at a dilution of 1 µg/mL in TBST supplemented with 1% skim milk powder and incubated overnight at 4C on a rocking platform, followed by washing and incubation with a goat anti-rabbit IgG horse radish peroxidase (HRP) conjugated secondary antibody at a dilution of 1:10,000 in TBST for 1 hr at room temperature. Chemiluminescent detection was performed using Pierce ECL Western blotting substrate (Product # 32209). Data courtesy of Dr. James Nagy's lab.

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Experimental details: Western blot analysis of Connexin43 (Cx43) was performed by loading 20 µg of mouse thalamus (not boiled) and 7 µL of molecular weight protein ladder per well onto a 10% Tris-Hcl polyacrylamide gel. Proteins were transferred to a PVDF membrane, and blocked in blocking buffer composed of 20 mM Tris-HCl pH 8, 150 mM NaCl and 0.2% Tween-20 (TBST) supplemented with 5% skim milk powder (Carnation) for 1 hr at room temperature. Cx43 was detected at ~43 kDa using an anti-Cx43 rabbit polyclonal antibody (Product # 71-0700) at a dilution of 1 µg/mL in TBST supplemented with 1% skim milk powder and incubated overnight at 4C on a rocking platform, followed by washing and incubation with a goat anti-rabbit IgG horse radish peroxidase (HRP) conjugated secondary antibody at a dilution of 1:10,000 in TBST for 1 hr at room temperature. Chemiluminescent detection was performed using Pierce ECL Western blotting substrate (Product # 32209).

Supportive validation

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Experimental details: Western blot was performed using Anti-Connexin 43 Polyclonal Antibody (Product # 71-0700) and a 43 kDa band corresponding to Connexin 43 was observed across the panel tested except in LNCaP which is reported to be negative. Scraped Whole cell extracts (50 µg lysate) of MDA-MB-231 (Lane 1), MCF-7 (Lane 2), T-47D (Lane 3), PC-3 (Lane 4) and LNCaP (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:10000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band (*) was also observed in a few cell lines.

Supportive validation

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Experimental details: Immunohistochemical Staining of human heart tissue using Rabbit anti Connexin-43 Polyclonal Antibody (Product # 71-0700)

Supportive validation

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Experimental details: Immunofluorescence analysis of Connexin 43 was performed on sections of adult heart. Tissue sections on slides were probed for 24 h at 4°C in a humidified chamber with rabbit polyclonal anti-Cx43 (Product # 71-0700) at an antibody concentration of 1-2 µg/mL diluted in 50 mM Tris-HCl, pH 7.4, containing 1.5% NaCl, 0.3% Triton X-100 (TBST) and 4% normal goat serum. After overnight incubation, sections were washed extensively for 1 h in TBST, and detection of primary antibody was performed for 1.5 h at room temperature with AlexaFluor-488-conjugated donkey anti-rabbit diluted 1:600 in TBST. Sections were then washed in TBST, then in TBS (without triton) and then coversliped with anti-fade medium. Images were taken on a Zeiss Z2 scanning microscope at x40 objective magnification, and show immunofluorescence labelling of Cx43 localized at gap junctions between cardiac myocytes at intercalated discs in adult mouse heart. Data courtesy of Dr. James Nagy's lab.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Connexin 43 was performed on sections of adult mouse thalamus. Tissue sections on slides were probed for 24 h at 4°C in a humidified chamber with rabbit polyclonal anti-Cx43 (Product # 71-0700) at an antibody concentration of 1-2 µg/mL diluted in 50 mM Tris-HCl, pH 7.4, containing 1.5% NaCl, 0.3% Triton X-100 (TBST) and 4% normal goat serum. After overnight incubation, sections were washed extensively for 1 h in TBST, and detection of primary antibody was performed for 1.5 h at room temperature with AlexaFluor-488-conjugated donkey anti-rabbit diluted 1:600 in TBST. Sections were then washed in TBST, then in TBS (without triton) and then coversliped with anti-fade medium. Images were taken on a Zeiss Z2 scanning microscope at x40 objective magnification, and show immunofluorescence labelling of Cx43 localized at gap junctions between cardiac myocytes at intercalated discs in adult mouse thalamus. Data courtesy of Dr. James Nagy's lab.

Supportive validation

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Experimental details: Immunohistochemistry analysis of CONNEXIN 43 showing staining in the cytoplasm of paraffin-embedded human heart 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 43 Rabbit Polyclonal Antibody (Product # 71-0700) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC 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 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

Supportive validation

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Experimental details: Figure 1 Atg16 associates with Cx43 in an early step of autophagosome biogenesis ( a ) Immunoblot for the indicated Cx of homogenates and autophagic vacuoles (AV) isolated from fed or 6h starved (Stv) mice. Quantification of enrichment is shown in Supplementary Fig. 1a . ( b ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 maintained in the presence or absence of serum for 4h. Single channels are shown in reverse black and white and magnification of single and merged images are shown in color. Arrows: colocalization of Atg16/Cx43 (yellow) or Atg16/Cx43/LC3 (white). Quantification is shown in Supplementary Fig. 1c . ( c ) Immunofluorescence for the indicated endogenous proteins in NRK cells maintained in serum-supplemented media without additions (None) or treated with chloroquine (CQ) for 4h. Full fields are shown in Supplementary Fig. 1d . ( d ) Maximum projection confocal microscopy 3D reconstruction (left) followed by a single plane time-lapse acquisition of the region outlined by the white square in HeLa cells expressing EBFP2-Cx43 (pseudocolored in red) and sfGFP-ATG16. ( e ) Immunofluorescence for LC3 and Atg16 in NRK cells expressing GFP-Cx43 but knocked down for Eps15. Single channels are shown in reverse black and white and merged images in color. Inset shows higher magnification. Bottom: Quantification of colocalization between Atg16 and Cx43 (n=3 wells, 4 independent experiments, >20 cells per experiment). Values are mean+s.e.m. (*) p

Supportive validation

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Experimental details: Figure 5 Internalized Cx43 is targeted to recycling endosomes during serum deprivation ( a-c ) Immunofluorescence for the indicated endosomal markers in NRK cells expressing GFP-Cx43 maintained in presence of serum (a ), absence of serum ( b ) or treated with lindane ( c ). Single black and white inverted channels, merged channels and higher magnification insets are shown. ( d ) Immunoblots for indicated proteins in isolated fractions from starved mouse liver HOM: homogenate; LE: late endosomes; CYT: cytosol; APG: autophagosomes; APL: autophagolysosomes. ( e ) Immunoblots for different connexins in homogenates and late endosomes isolated from fed and 6h starved mice. ( f-h ) Fluorescence images of NRK cells expressing GFP-Cx43 maintained in presence of serum (f ), absence of serum ( g ) or treated with lindane ( h ) and incubated with Alexa594-transferrin for 15 minutes before fixation. Top: single and merged channels and boxed area at higher magnification. Bottom: 3D reconstructions and higher magnification details of the boxed areas. Nuclei are highlighted with DAPI. Bars: 5 mum. Uncropped images of blots are shown in Supplementary Fig. 9 .

Supportive validation

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Experimental details: Figure 7 Cx43 modulates formation of pre-autophagosomal structures through direct interaction with autophagy-related proteins ( a ) Co-immunostaining for Cx43 and the indicated Atgs in NRK cells maintained in the presence or absence of serum (top). Single and merged channels of the 3D reconstruction of the boxed regions are shown (bottom). Nuclei are highlighted with DAPI. ( b, c ) Immunoblot for the indicated proteins of immunoprecipitates (IP) of Cx43 in NRK cells maintained in the presence or absence of serum ( b ) or treated or not with 3-methyladenine ( c ) Atg5 is shown as negative control for IP. ( d ) Co-staining for Atg9 and e-cadherin in NRK cells control and knocked down for Cx43. Single and merged channels at higher magnification areas are shown. ( e ) Immunofluorescence for endogenous Atg9 and Cx43 in NRK cells control or knocked down for Eps15. ( f, g ) Immunofluorescence for endogenous Cx43 (green) in NRK cells control or knocked down for Atg14 ( f ) or Atg9 ( g ) maintained in the presence or absence of serum. ( h ) Immunofluorescence of endogenous Cx43 in NRK cells control or knocked down for Atg14 treated (or not) with Tx or Lnd (left). e-cadherin staining (red) is shown to highlight plasma membrane in g and h . Full fields are shown in Supplementary Fig. 6h . Quantification of intracytoplasmic Cx43-positives vesicles (right) (n= 3 wells, 3 independent experiments, >30 cells per experiment). Values are mean+s.e.m and significant for (*) p

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Experimental details: Figure 8 Depletion of connexins induces autophagosome formation ( a ) Immunoblot for the indicated proteins in mouse embryonic fibroblasts (MEFs) control or knocked down (-) for the three different connexins. ( b ) Immunofluorescence of LC3 in MEFs control (Ctr) or knocked down (-) for three different Cx maintained in the presence or absence of serum. Left: representative fields and higher magnification insets. Nuclei are highlighted with DAPI. Right: Quantification of the average number of LC3-positive puncta per cell (n= 3 wells, 3 independent experiments, >50 cells per experiment). (c) Immunoblot for LC3 in the same cells maintained in the presence of serum untreated (-) or treated with inhibitors of lysosomal proteases (PI) for the indicated times. GAPDH is shown as loading control. ( d ) Representative images of MEFs control or knocked-down (-) for the indicated proteins transfected with the mCherry-GFP-LC3 tandem reporter. Right: quantification of the average number per cell of puncta positive for GFP and mCherry (autophagosomes; APG) and those positive only for mCherry (autophagolysosomes; APL) (n= 3 wells, 3 independent experiments, >50 cells per experiment). ( e ) Electron micrographs of MEFs control or knock-down for the indicated Cx. Insets show autophagic vesicles. Bottom: Quantification of the average number of AV (left) and percentage of cytosolic area (right) occupied by AVs (n= 4 wells, 4 independent experiments, >10 micrographs per well). ( f ) Immunoblot for

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Experimental details: Figure 3 Immunohistochemical staining of Connexin 43 at different stages of pregnancy . (A) Non-pregnant (group 1), (B) term-pregnant (group 2), (C) in normal labour (group 3), D in prolonged labour (group 4), (E) in normal labour, magnification 1 x 400 with oil, (F) negative control. Magnification in A, B, C, D and F is 1 x 200 Positive staining is brown. The grading of staining is presented in Table 3.

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Experimental details: Figure 4 Co-localization of Connexin 43 and Syndecan 3 in uterine tissue during normal labour . Sections from uterine tissue from women in normal labour (panel A-C), patients at term (panel D-F) and patients with prolonged labour (panel G-I) were obtained and prepared as described above. A monoclonal antibody against syndecan 3 was added, followed by Alexa Fluor 633 goat anti-rabbit IgG antibody. After that, a Connexin 43 antibody followed by Alexa Fluor 488 rabbit anti-mouse IgG antibody was added. The excitation for Connexin 43 (panel A, D and G) and Syndecan 3 (panel B, E and H) are shown. The merged picture demonstrates the co-localization in the tissue (panel C, F and I).

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Experimental details: Fig 3 Time-dependent and rapid decrease in Cx43 protein levels in ethanol-treated HL-1 cells. (A-D) Cells were incubated in medium containing 2% ethanol for the indicated time periods, and the protein levels of Cx43, Cx40 and Cx45 were determined by western blot analysis (A). Hsc70 served as a loading control. Quantitative analysis of Cx43 (B), Cx40 (C) and Cx45 (D) is shown. Each graph shows mean+-S.E. (n = 4-6). **, p

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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 2 Isolation and characterization of cell sheets from c-kit+ CPC. (a) CPC sheets formed after 3 days of culture detached at room temperature onto thermoresponsive surfaces yielding a scaffold-free monolayered cell sheet. (b) Hematoxylin-eosin staining shows that the cell sheet represents a well-organized structure. CPC in the cell sheets survived, retained the ability to proliferate (Ki67), expressed progenitor cell marker Gata 4, interconnected with each other via connexin-43 gap junctions, and were surrounded extracellular matrix proteins (collagen 1, collagen 3, and fibronectin). (c) Macroscopic visualization of epicardially attached CPC sheet after implantation.