- 36-4700 - Invitrogen Antibodies GJC2 antibody

Submitted references Microtubule-assisted altered trafficking of astrocytic gap junction protein connexin 43 is associated with depletion of connexin 47 during mouse hepatitis virus infection.

Basu R, Bose A, Thomas D, Das Sarma J
The Journal of biological chemistry 2017 Sep 8;292(36):14747-14763

Defective lymphatic valve development and chylothorax in mice with a lymphatic-specific deletion of Connexin43.

Munger SJ, Davis MJ, Simon AM
Developmental biology 2017 Jan 15;421(2):204-218

Segregated Foxc2, NFATc1 and Connexin expression at normal developing venous valves, and Connexin-specific differences in the valve phenotypes of Cx37, Cx43, and Cx47 knockout mice.

Munger SJ, Geng X, Srinivasan RS, Witte MH, Paul DL, Simon AM
Developmental biology 2016 Apr 15;412(2):173-90

Absence of venous valves in mice lacking Connexin37.

Munger SJ, Kanady JD, Simon AM
Developmental biology 2013 Jan 15;373(2):338-48

Disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis.

Markoullis K, Sargiannidou I, Gardner C, Hadjisavvas A, Reynolds R, Kleopa KA
Glia 2012 Jul;60(7):1053-66

Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax.

Kanady JD, Dellinger MT, Munger SJ, Witte MH, Simon AM
Developmental biology 2011 Jun 15;354(2):253-66

Pathologic and phenotypic alterations in a mouse expressing a connexin47 missense mutation that causes Pelizaeus-Merzbacher-like disease in humans.

Tress O, Maglione M, Zlomuzica A, May D, Dicke N, Degen J, Dere E, Kettenmann H, Hartmann D, Willecke K
PLoS genetics 2011 Jul;7(7):e1002146

Ablation of connexin30 in transgenic mice alters expression patterns of connexin26 and connexin32 in glial cells and leptomeninges.

Lynn BD, Tress O, May D, Willecke K, Nagy JI
The European journal of neuroscience 2011 Dec;34(11):1783-93

Ablation of Cx47 in transgenic mice leads to the loss of MUPP1, ZONAB and multiple connexins at oligodendrocyte-astrocyte gap junctions.

Li X, Penes M, Odermatt B, Willecke K, Nagy JI
The European journal of neuroscience 2008 Oct;28(8):1503-17

Identification of connexin36 in gap junctions between neurons in rodent locus coeruleus.

Rash JE, Olson CO, Davidson KG, Yasumura T, Kamasawa N, Nagy JI
Neuroscience 2007 Jul 29;147(4):938-56

Connexin-47 and connexin-32 in gap junctions of oligodendrocyte somata, myelin sheaths, paranodal loops and Schmidt-Lanterman incisures: implications for ionic homeostasis and potassium siphoning.

Kamasawa N, Sik A, Morita M, Yasumura T, Davidson KG, Nagy JI, Rash JE
Neuroscience 2005;136(1):65-86

Expression of zonula occludens-1 (ZO-1) and the transcription factor ZO-1-associated nucleic acid-binding protein (ZONAB)-MsY3 in glial cells and colocalization at oligodendrocyte and astrocyte gap junctions in mouse brain.

Penes MC, Li X, Nagy JI
The European journal of neuroscience 2005 Jul;22(2):404-18

Connexin47, connexin29 and connexin32 co-expression in oligodendrocytes and Cx47 association with zonula occludens-1 (ZO-1) in mouse brain.

Li X, Ionescu AV, Lynn BD, Lu S, Kamasawa N, Morita M, Davidson KG, Yasumura T, Rash JE, Nagy JI
Neuroscience 2004;126(3):611-30

Coupling of astrocyte connexins Cx26, Cx30, Cx43 to oligodendrocyte Cx29, Cx32, Cx47: Implications from normal and connexin32 knockout mice.

Nagy JI, Ionescu AV, Lynn BD, Rash JE
Glia 2003 Dec;44(3):205-18

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of mouse brain homogenates using Rb anti-Connexin 47 (Product # 36-4700).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Connexin 47 showing staining in the membrane and cytoplasm of paraffin-embedded mouse cerebellum 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 47 polyclonal antibody (Product # 36-4700) 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.

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Experimental details: Immunofluorescence analysis of Connexin47 (Cx47) was performed on sections of adult mouse brain. Tissue sections on slides were probed for 24 h at 4°C in a humidified chamber with rabbit polyclonal anti-Cx47 (Product # 36-4700) 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 Cx47 localized at gap junctions between astrocyte processes and the cell bodies and initial processes of oligodendrocytes in the thalamus of adult mouse brain. Data courtesy of Dr. James Nagy's lab.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Connexin 47 showing staining in the membrane and cytoplasm of paraffin-embedded human brain 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 47 polyclonal antibody (Product # 36-4700) 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 47 showing staining in the membrane and cytoplasm of paraffin-embedded mouse cerebellum 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 47 polyclonal antibody (Product # 36-4700) 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.

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Submitted by: Invitrogen Antibodies (provider)
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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Supportive validation

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Experimental details: Figure 1 Generation of mCx47 M282T mice with LacZ reporter gene. A, Scheme of homologous recombination of the targeting vector into the Cx47 coding region. The resulting transgenic allele ( Cx47M282Tneo ) includes mCx47 M282T coding DNA, an internal ribosome entry site (IRES) followed by a nuclear localization signal (nls) fused to LacZ coding DNA and a neomycin selection cassette flanked by frt-sites. The endonuclease Bsm BI restriction site was generated by T to C transition on nucleotide 845 of the Gjc2 coding region, resulting in the mutant mCx47 M282T . B, Flp recombinase activity causes deletion of the neomycin selection cassette. Specific primer binding sites and Mfe I restriction sites are indicated. C, PCR products specific for wild-type (570 bp) and transgenic loci (730 bp) using DNA obtained from tail tip tissue. D, Presence of the mutant allele was proven by PCR amplification of a 700 bp Cx47 fragment and subsequent Bsm BI digestion. Only mutant alleles yielded the 350 bp fragment. E, PCR analysis resulted in amplification of the 1,700 bp fragment of the neomycin selection cassette deprived allele and the 450 bp fragment of the Cx47M282Tneo locus. F, Homologous recombination and different allelic combinations were confirmed by Southern blot hybridization using a probe derived from the 3' region of Cx47. Mfe I digestion of genomic DNA prepared from liver resulted in the of 4.6 kb fragment for the Cx47 wildtype allele, the 10.5 kb fragment for the Cx47M282Tneo

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Experimental details: Figure 10 Expression of mutant mCx47 M282T results in delayed oligodendrocytic differentiation. Immunoblots were performed with cerebellar lysates. At least three animals per group were analyzed. For each oligodendrocytic marker, protein levels were normalized to beta-tubulin expression as internal standard. Expression levels of Cx47 +/+ mice were set to 100% per group. A, Densitometric quantification of immunoblot analyses revealed significantly decreased CNPase expression in P10, P16 and P40 Cx47 M282T/M282T mice compared to their wildtype littermates (** p

Supportive validation

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Experimental details: Figure 2 mCx47 M282T and beta-gal expression in oligodendrocytes of 40-day-old mutant mice. A-C, LacZ staining of 50 um brain slices obtained from Cx47 +/M282T mice. The beta-gal activity is restricted to nuclear localisation. A, beta-Gal positive cells in the corpus callosum show typical oligodendrocytic chain-like organization and coexpression of the oligodendrocytic marker CNPase. LacZ expression was not detectable in GFAP-positive astrocytes (B) or NeuN-positive neurons (C). D, Immunostaining with Cx47 antibodies (green) revealed signals in close proximity of Syto-61 stained nuclei in the corpus callosum of Cx47 +/+ mice. Weaker signals were also found more distal to the nuclei. E, Cx47 antibody stainings on heterozygous Cx47 +/M282T mice resulted in weaker, but similarly localized immunosignals compared to those obtained on wildtype brain tissue. Cx47 gap junction immunosignals were predominantly localized close to beta-gal positive nuclei indicated by antibody staining. F, Homozygous Cx47 M282T/M282T mice showed apparent beta-gal immunoreactivity, but robust Cx47 gap junction immunosignals were not detected in the perikarya. Very weak Cx47 signals were noticed in brain tissue of Cx47 M282T/M282T mice. G, Immunoblot staining against beta-gal on whole brain lysates of 40-day-old mice yielded strong signals with Cx47 M282T/M282T , weaker signals with Cx47 +/M282T and no signals with Cx47 +/+ tissue. H, Immunoblot analysis on Cx47 protein resulted in unspecific bands at 50

36-4700

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