13-8200

antibody from Invitrogen Antibodies

Targeting: GJB1 CMTX, CMTX1, CX32

Provider product page for 13-8200

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Antibody data

Antibody Data
Antigen structure
References [41]
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Validations
Other assay [17]

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Product number: 13-8200 - Provider product page
Provider: Invitrogen Antibodies
Product name: Connexin 32 Monoclonal Antibody (CX-2C2)
Antibody type: Monoclonal
Antigen: Synthetic peptide
Reactivity: Human, Mouse, Rat
Host: Mouse
Isotype: IgG
Antibody clone number: CX-2C2
Vial size: 100 µg
Concentration: 0.5 mg/mL
Storage: -20°C

GJB1 protein structure - 13-8200 shown in red.

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: NULL

Supportive validation

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Experimental details: NULL

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 1 Nf1 Loss or HRas Activation in Oligodendrocytes Causes Optic-Nerve Enlargement, Myelin Decompaction, Loss of Claudin-11, and Altered Cx32 Localization (A) Gross micrographs of optic nerves at the level of the chiasm in 12-month-old animals. Ruler shows 1 mm markings. (B) Toluidine-blue-stained semithin optic-nerve cross-sections, 1 mm rostral to the chiasm. (C) Optic-nerve g-ratio scatterplots (~5,000 axons/graph; measurements 1 mm rostral to the chiasm in three to five electron micrographs at 10,000x). Red box: 99% of WT g-ratios superimposed upon graphs of other genotypes. ANOVA, Tukey post hoc, p = 2.2 x 10 -16 . (D) Diagram of myelin compaction. Brown circle: axon cross-section; gray: major dense lines (MDLs); orange: Cx32 GJs between compact myelin layers; blue lines: claudin-11+ TJs in compact myelin. (E) Electron micrographs (scale bar, 10 nm) of WT, PLPCre;Nf1 fl / fl , and CNP-HRas optic-nerve myelin. White arrows: intraperiod lines (ILs); black arrows: MDLs. (F-I) Western blots of total optic-nerve lysate or whole-brain cytosolic, endosomal, or myelin fractions showing myelin proteins (F), claudin-11 (G), or Cx32 cellular localization (H and I). n = 3-5 animals/genotype/experiment. See also Figures S1, S2, S3, and S4 .

Supportive validation

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Experimental details: Fig 8 Connexin 32 expression in HK-2, HPT, and HK-2 cells expressing MT3. Messenger RNA of connexin 32 was assessed with real-time PCR and expressed as a fold increase of the HK-2 cells stably transfected with the blank vector. The change in connexin 32 expression was normalized to the change in beta-actin expression. Western analysis of connexin 32 is shown below the graph.

Supportive validation

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Experimental details: Fig 11 Effect of altered domains of metallothionein-3 on the expression of connexin 32 in HK-2 cells. A) Messenger RNA of connexin 32 assessed with real-time PCR and expressed as fold change in expression versus HK-2 cells transfected with the blank vector. The change in connexin 32 expression was normalized to the change in beta-actin expression. Significant differences from HK-2 (BV) are designated as *** p < 0.0001. B) Western analysis of connexin 32 was conducted in identical cultures as in (A). beta-actin was used as a loading control and for densitometric normalization. Densitometry is shown beside the corresponding blot.

Supportive validation

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Experimental details: Figure 4 SW620 cell-secreted factors overexpress the endothelial Cx32 favoring tubulogenesis A. Endothelial cell localization of Cx32 in CRC cell-conditioned media. HMEC were stimulated with SW480-CM or SW620-CM for 6 h and double-stained for ZO-1 and Cx32. Representative micrographs showing the strong labelling of Cx32 induced by SW620-CM and the combined image of co-localization with ZO-1 (yellow); DAPI staining of nuclei ( n = 3, bar 20 mum). B. SW620-CM increase the Cx32 expression in HMEC. A higher Cx32 protein level was detected in response to SW620-CM compared with SW480-CM by immune-blot analysis (no cell expression in unstimulated HMEC). Representative of 5 experiments (Hsc70 as loading control; 150 mug/lane). C-D. In vitro tubulogenesis assay of HMEC pretreated or not (control IgG) with inhibitory monoclonal antibody against Cx32 (anti-Cx32 mAb). HMEC were plated on Matrigel-coated 24-well plates, incubated with SW620-CM for 6 h, and photographed. C. Representative photos of tube formation in HMEC intracellularly delivered with 0.2 mug anti-Cx32 mAb or control IgG (Bar 80 mum). The dotted areas are enlarged in the inserts on the right. Arrows indicated branch points. D. Number of branch points per field of view was quantified (at least 80 single cells were scored; mean +- SD, n = 4; * P < 0.01 vs control).

Supportive validation

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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

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Figure 2. Immunostainings and fluorescence-activated cell sorting by GAP11. (A): Immunofluorescent staining with Cx32 and GAP11 antibodies (green) on frozen sections of human cornea and limbus with mouse liver as Cx32 positive control and PI counterstaining (red). (B): Flow cytometry and fluorescence-activated cell sorting of primary cultured human limbal epithelial cells by GAP11 (panels 2 and 3), with second Ab only as negative control (panel 1). A, negative cells; B, positive cells; C, selected negative cells (Cx43 dim ) with FITC intensity less than 0.2; D, selected positive cells (Cx43 bright ) with FITC intensity higher than 100. (C): Real-time PCR for Cx43 expression by ALL and selected Cx43 bright and Cx43 dim populations. Abbreviations: Ab, antibody; ALL, unfractioned; Cx, connexin; FITC, fluorescein isothiocyanate; PCR, polymerase chain reaction.