13-8300

antibody from Invitrogen Antibodies

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

Provider product page for 13-8300

Western blot

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Immunohistochemistry

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Antibody data

Antibody Data
Antigen structure
References [132]
Comments [0]
Validations
Western blot [4]
Immunohistochemistry [4]
Other assay [24]

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Product number: 13-8300 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 43 Monoclonal Antibody (CX-1B1)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Description: CX-1B1 is specific for one of the unphosphorylated forms of Connexin 43. This antibody was at one time thought to be specific for all unphosphorlyated forms of Connexin 43, but that can no longer be claimed since it was discovered that Connexin 43 can be phosphorylated at multiple sites. Recent studies have shown that phosphorylation occurs at a serine within the epitope recognized by this antibody. CX-1B1 recognizes Connexin 43 only when the serine at residue 368 is unphosphorylated. CX-1B1 will, however, recognize Connexin 43 if it is phosphorylated at sites besides Ser368. In some cell types, cross-reactivity with a ~70 kDa protein of unknown identity has been observed. This cross-reactivity is likely due to a shared epitope. Reactivity has been confirmed by western blot analysis of extracts derived from dog heart, human heart, mouse brain, mouse heart, rat brain, and Clone 9 (C1-9) rat liver cells. Clone 9 is the recommended positive control for CX-1B1. This antibody has also been used in immunofluorescence applications and immunolocalization of Connexin 43, however, for immunohistochemical applications polyclonal Rabbit anti-Connexin 43 (Cat. No. 71-0700) is recommended.
Reactivity: Human, Mouse, Rat, Canine
Host: Mouse
Isotype: IgG
Antibody clone number: CX-1B1
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

GJA1 protein structure - 13-8300 shown in red.

Supportive validation

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane 1), C2C12 (Lane 2), Caco-2 (Lane 3), HUVEC (Lane 4) and HAEC (Lane 5). The blots were probed with Anti-Connexin 43 Mouse Monoclonal Antibody (Product # 13-8300, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A ~ 50 kDa band corresponding to Connexin 43 was observed across cell lines tested except Caco-2. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane 1), C2C12 (Lane 2), Caco-2 (Lane 3), HUVEC (Lane 4) and HAEC (Lane 5). The blots were probed with Anti-Connexin 43 Mouse Monoclonal Antibody (Product # 13-8300, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A ~ 50 kDa band corresponding to Connexin 43 was observed across cell lines tested except Caco-2. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of NTERA-2 (Lane 1), C2C12 (Lane 2), Caco-2 (Lane 3), HUVEC (Lane 4) and HAEC (Lane 5). The blots were probed with Anti-Connexin 43 Mouse Monoclonal Antibody (Product # 13-8300, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A ~ 50 kDa band corresponding to Connexin 43 was observed across cell lines tested except Caco-2. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 12 % Bis-Tris gel (Product # NP0342BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® 2 Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Knockout of Connexin 43 was achieved by CRISPR-Cas9 genome editing. Western blot analysis of Connexin 43 was performed by loading 20 µg of HeLa wild type (Lane 1), HeLa Cas9 control (Lane 2), HeLa Connexin 43 knockout (Lane 3) whole cell extracts. The blot was probed with Anti-Connexin 43 Monoclonal Antibody (CX-1B1) (Product # 13-8300) using 2µg/mL and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177)using 1:4000 dilution. Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to Connexin 43.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Connexin 43 showing staining in the membrane of paraffin-embedded human lung adenocarcinoma (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 monoclonal antibody (Product # 13-8300) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Connexin 43 showing staining in the membrane of paraffin-embedded mouse 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 monoclonal antibody (Product # 13-8300) diluted in 3% BSA-PBS at a dilution of 1:100 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Connexin 43 showing staining in the membrane of paraffin-embedded mouse testis 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 monoclonal antibody (Product # 13-8300) 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.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Connexin43 (Cx43) was performed on sections of adult brain. Tissue sections on slides were probed for 24 h at 4°C in a humidified chamber with mouse monoclonal anti-Cx43 (Product # 13-8300) 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-mouse 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 astrocytes in the thalamus of adult mouse brain. Data courtesy of Dr. James Nagy's lab.

Supportive validation

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Supportive validation

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Experimental details: Figure 1 Upregulation of connexin-43 in tumor cell-endothelial cell contact areas in vitro and in vivo . (a)-(d) Immunocytofluorescence detecting connexin-43 (Cx43; red) in co-culture of 4T1-GFP cells (green cytoplasmic staining with blue nuclei) and pulmonary microvascular endothelial cells (PMVECs; blue nuclei without cytoplasmic staining). 4T1-GFP and PMVECs were plated together in co-culture and examined under the green fluorescent protein (GFP) channel (4T1 cells), UV-channel (nuclei), and Cy5-channels (Cx43), and shown as an overlay of all channels indicating the location of 4T1-GFP cells (green), nuclei of both cell types (blue), and Cx43 (red). In (a), (b), and (c), upregulation of Cx43 expression (red) is seen at the contact areas between the tumor cells and PMVECs (white arrows). Cells not in a heterologous contact show little expression of Cx43 (yellow arrows). Panel (d) shows an additional overlay of the DIC image to further emphasize the Cx43 intense expression at the areas of contacts (white arrow) between an endothelial cell on the left and a 4T1-GFP cell on the right. (e) Immunohistofluorescence of a lung metastatic tumor section stained for both Cx43 (red) and CD31 (green) with nuclear DAPI staining in blue. Most endothelial cells within the tumor mass (green) showed an increase in Cx43 staining (arrows). (f) Immunohistofluorescence of a fixed mouse lung section after 12 hours of tail vein injection of tumor cells. Autofluorescence of lung tissue in the GFP-c

Supportive validation

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Experimental details: Figure 2 Connexin-43 expression marks the sites of micrometastases . (a)-(d), (h) Immunohistofluorescence fixed sections of mouse lung after 12 hours of tail vein injection of 4T1-GFP cells or connexin-43 (Cx43) mutant variants C61S, G138R, or Cx43OE. Autofluorescence of lung tissue and 4T1-GFP fluorescence (green fluorescent protein (GFP) channel green color) and Cx43 signal (Cy5-channel red color), and nuclear staining (DAPI channel blue color) were overlaid. Panels (a)-(d) show images of lung sections taken at low magnification. Panel (a) shows the sections from a lung after tail vein injection of control 4T1-GFP tumor cells. Cx43 (red) was upregulated in vessels containing tumor cells (yellow arrow), while vessels not containing tumor cells from the same lung had barely detectable Cx43 signal (white arrows). Lung sections with C61S expressing tumor cells exhibited similar pattern of Cx43 expression (b), whereas those with the dominant-negative G138R expressing cells exhibited decreased staining of Cx43 (c). In contrast, in lung sections with Cx43 overexpressing tumor cells, a significant increase in the number of intravascular tumor cells, an increase in the number of vessels containing adherent tumor cells, and induction of Cx43 was seen (d). Most of the vessels with Cx43 staining appeared to contain tumor cells (yellow arrows). Panels (e)-(h) show higher-magnification images of lung sections containing tumor cells overexpressing Cx43. Panel (e) shows a DIC image of a ve

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 6 Atg14 is recruited to Cx43-enriched plasma membrane regions during nutritional deprivation ( a ) Immunoblot for the indicated proteins in homogenates and purified plasma membrane from fed (F) and 24 h starved mice (Stv). Starvation-induced changes in the PM content relative to fed levels were as follows: Vps15 (1.2+0.2), Beclin-1 (0.7+0.5), Vps34 (0.9+0.1), Atg16 (1.1+0.3), Atg14 (2.5+0.2) and only Atg14 were significant for p

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

Supportive validation

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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

Supportive validation

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Experimental details: Figure 5 Shared expression of progenitor markers in atrial and bone marrow-derived PBCs. PBCs were collected from either (a-e) atrial cultures or (f-j) BIX01294 plus CCM-treated MSCs and immunostained following (a-c; e-h, i) their attachment on Cell-Tak coated chamber slides or (d, i) being cytospun onto glass slides. Subsequent immunofluorescent labeling of the cells (shown in gray) was used to detect the expression of (a, f) Sca-1 (b, g) CD90 (c, h) Isl-1, (d, i) Cx43, and (e, j) Cx40. Cultures were counterstained with DAPI to visualize nuclei (blue). Scale bar = 25 mu m.

Supportive validation

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Experimental details: Figure 6 Expression of connexin-43 by the mutant variants . (A) Immunocytofluorescence analysis of connexin-43 (Cx43) expression in wild-type 4T1-GFP tumor cells and in 4T1-GFP cells transfected with expression vectors of the mutant G138R and C61S in addition to a vector expressing two copies of wild-type Cx43 (Cx43OE). Endogenous Cx43 in wild-type 4T1-GFP cells is present mostly at the plasma membrane between the cells (3A, 4T1, white arrows). The G138R dominant-negative mutant was expressed both at the plasma membrane (white arrows) and in the cytoplasm (yellow arrow). Wild-type overexpression led to upregulation of Cx43 in both the plasma membrane (white arrows) and in the cytoplasm (yellow arrows, 3A, Cx43OE). Cx43 with the C61S mutation was mostly in the cytoplasm (yellow arrows, 3A, C61S). (B) Quantification of the immunocytofluorescence signal of Cx43 in 4T1-GFP cells expressing mutant and wild-type Cx43. All of the transfectants showed at least a factor or two more Cx43 expression compared with endogenous expression (control) or with mock transfection. (C) Western blot analysis showing increased expression of Cx43 forms in transfected cells when compared with non-transfected 4T1-GFP cells. The quantification of bands from three blots illustrates that the transfected cells show an at least two-fold increase in Cx43 expression compared with wild-type 4T1-GFP cells. * P < 0.05.