GJA1 Antibody
Product Specs
Target Background
- LB2003 cells, lacking three essential potassium uptake transport mechanisms, are unable to proliferate in low potassium medium. However, the expression of Cx26, Cx43, or Cx46 rescues their growth defect, demonstrating growth complementation. (PMID: 27789753)
- A novel role for Cx43-formed unidirectional gap junctional intercellular communication was identified in mediating metabolic coupling between cancer-associated fibroblasts and non-small cell lung cancer cells. This interaction facilitates malignant progression of NSCLC by enhancing oxidative phosphorylation and increasing ATP-activated PI3K/Akt and MAPK/ERK signaling pathways. (PMID: 30453281)
- A study demonstrated overexpression of Ubc9 protein in osteosarcoma. Silencing Ubc9 in osteosarcoma cell lines induced decoupling of SUMO1 from Cx43, leading to increased free Cx43 levels. This is crucial for reconstructing gap junction intercellular communication and recovering cellular functions. (PMID: 29956745)
- These results indicate that the Cx43 SH3-binding domain, along with the CT9 region, critically controls hemichannel activity at high intracellular calcium concentrations, which may be involved in pathological hemichannel opening. (PMID: 29218600)
- Pinocembrin alleviated ventricular arrhythmia in ischemic/reperfusion rats by enhancing Na+-K+ATPase and Ca+-Mg2+ATPase activity and upregulating Cx43 and Kir2.1 protein expression. (PMID: 30022020)
- A Tunisian family with Oculodentodigital Dysplasia (ODDD) exhibited neurologic signs with anticipation, an uncommon feature of this disease. This study extends the mutational spectrum of the GJA1 gene by identifying a novel mutation in the L2 region of Cx43. (PMID: 30204976)
- Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43). This review summarizes current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43-mediated effects. (PMID: 29748463)
- In progesterone control of myometrial contractility during pregnancy and labor, while liganded nuclear progesterone receptor B can suppress the expression of Cx43, unliganded progesterone receptor A paradoxically translocates to the nucleus where it acts as a transcriptional activator of this labor gene. (PMID: 27220952)
- Ezrin-anchored PKA phosphorylates serine 369 and 373 on connexin 43 to enhance gap junction assembly, communication, and cell fusion. (PMID: 29259079)
- A study found a significant difference in the expression of Cx43 and SUMO1 between cancer stem cells and non-cancer stem cells in liver cancer. Co-expression of Cx43 and SUMO1 in cancer stem cells significantly improved gap junction intercellular communication. (PMID: 29393359)
- The frequency of the single nucleotide polymorphism rs2071166 was significantly higher in atrial septal defect cases than in healthy controls. The CC genotype at rs2071166 site in Cx43 was correlated with an increased risk for atrial septal defect, and the C allele was positively correlated with this condition. (PMID: 29198211)
- Inhibition of Connexin43 signaling plays a more significant role in regulating cell proliferation than cell migration. (PMID: 29463027)
- Our results suggest that keratinization in the hair follicle is closely related to the decrease in Cx43 expression. (PMID: 28960405)
- Human Cx46 V44M mutant causing cataracts results in abnormally decreased formation of gap junction plaques and impaired gap junction channel function. (PMID: 29321356)
- Abnormal expression of Cx43 in the cerebral arteries may play a significant role in the formation of vascular intima thickening in patients with moyamoya disease. (PMID: 29395647)
- Findings demonstrate how SRC3 and Cx43 regulation between bone marrow stromal cells (BMSCs) and myeloma cells mediate cell growth and disease progression. (PMID: 29075794)
- Mutations of known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373 were generated. S365A, S365E, S368A, S368E, and S373A mutants bound ZO-1 throughout the GJ plaques, while the S373E mutant did not bind ZO-1 at all. These results suggest that 1) S365 and S373 phosphorylation promote forward trafficking, and 2) phosphorylation on these residues appears to prevent premature binding of ZO-1. (PMID: 29021339)
- These data suggest that chronic exposure to glucose-evoked TGFbeta1 induces an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. (PMID: 29587265)
- Cx43, a transmembrane protein initially described as a gap junction protein, participates in all forms of communication, including extracellular vesicles, tunneling nanotubes, or gap junctions. (Review) (PMID: 29025971)
- One novel homozygous variant c.169C>T and one heterozygous SNP c.624C>T (rs530633057) were identified in 124 SUNDS cases (one case for each detected variant) and none of the 125 healthy controls. This is the first report of GJA1 gene variations in SUNDS in the Chinese Han population, suggesting a novel susceptibility gene for Chinese sudden unexplained nocturnal death syndrome. (PMID: 27992820)
- The functional modulation of connexin 43 (Cx43) indicates its involvement in olfactory ensheathing cells-conditioned medium (OEC-CM) mediated neuroprotection. (PMID: 28488330)
- To determine the role of connexin43 hemichannels in diabetic retinopathy, changes in cytokine and ATP release were evaluated after treatment with Peptide5, a connexin43 hemichannel blocker. Co-application of glucose and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF, and ATP. Peptide 5 blocked this effect and prevented ATP release, indicating a role for connexin-43 hemichannels. (PMID: 29158134)
- Human Cx40/Cx45 and Cx43/Cx45 heterotypic gap junctions were investigated by recombinant expression in GJ-deficient cells. (PMID: 28760564)
- The results of this study show that total (whole-cell) Cx43, but not Cx30, protein levels are upregulated in the sclerotic hippocampus, both in human and experimental temporal lobe epilepsy. (PMID: 28795432)
- Data suggest that the level of CX43 expression in breast tumors is altered when compared to the normal tissue. While some reports show that its levels decrease, other evidence suggests that its levels are increased, and protein localization shifts from the plasma membrane to the cytoplasm. In either case, the prevailing theory is that breast tumor cells have reduced gap junction communication within primary tumors. [review] (PMID: 28902343)
- An oncogenic E3 ubiquitin ligase promotes loss of gap junctions and Cx43 degradation in human carcinoma cells. (PMID: 28733455)
- Administration of metformin can protect H9c2 cells against hyperglycemia-induced apoptosis and Cx43 down-regulation, in part, mediated through the induction of the autophagy pathway. (PMID: 28824303)
- DNA methylation of GJA-1 of human hippocampus and prefrontal cortex in major depression is unchanged in comparison to healthy individuals. (PMID: 28645745)
- HepaCAM associates with connexin 43, a main component of gap junctions, and enhances connexin 43 localization to the plasma membrane at cellular junctions. (PMID: 27819278)
- A region of CX43 (amino acids 266-283) exerts an important anti-tumor effect in patient-derived glioblastoma models that includes impairment of GSC migration and invasion. (PMID: 28712848)
- The low connexin 43 expression levels may reflect both a reduction in astroglial functional gap junctions and semicanals and a decrease in the amount of the protein itself, which has independent anti-oncogenic properties. (PMID: 28418351)
- Cx43 inhibited the growth of U251 cells, promoted morphological changes and migration, and inhibited apoptosis via a mitochondria-associated pathway. (PMID: 28615614)
- Macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of atrial fibrillation, probably by down-regulating the protein and gene expression of Cx43 via ERK1/2 kinase activation. (PMID: 28429502)
- These studies highlight the importance of Cx43 expression and function during osteoblast and chondrocyte differentiation. (PMID: 28177159)
- The observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/ERK2/Cx43 signaling axis at the interfaces between Cx43-high prostate cancer and endothelial cells. (PMID: 28396058)
- This review presents an overview of the key phosphatases known to interact with Cx43 or modulators of Cx43, as well as some possible therapeutic targets to regulate phosphatase activity in the heart. (PMID: 28478048)
- Many of the known non-canonical roles of Cx43 can be attributed to the recently identified six endogenous Cx43 truncated isoforms, which are produced by internal translation. In general, alternative translation is a new leading edge for proteome expansion and therapeutic drug development. (PMID: 28576298)
- Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics. (PMID: 28414037)
- This article explores the complex regulatory and signaling networks controlled by the Cx43 C-terminus, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. (PMID: 28526583)
- Cx43 plays a role in the regulation of the metastatic potential and migration of prostate cancer cells. (PMID: 28651025)
- Results showed that connexin 43 enhanced oxaliplatin cytotoxicity through gap junctional communication function, and high concentrations of oxaliplatin inhibited connexin 43 expression to counteract its cytotoxicity. (PMID: 28478804)
- Connexin 43 expression was significantly reduced or lost in prostate cancer tissues, which was associated with advanced clinicopathological features and poor biochemical recurrence-free survival of patients after radical prostatectomy. (PMID: 27623212)
- To match the stimulatory effect on acid uptake, cell-to-cell coupling in NHDF-Ad and CCD-112-CoN cells was strengthened with TGFbeta1. Importantly, the activities of stromal AE2 and connexin-43 do not place an energetic burden on cancer cells, allowing resources to be diverted for other activities. (PMID: 27543333)
- This study highlights the role of polyamines in the regulation of connexin 43 (Cx43) gap junctions. The study found that polyamines augment cell-to-cell communication and prevent uncoupling of Cx43 gap junctions induced by acidification and high intracellular calcium concentrations. (PMID: 28134630)
- Cx43 expression, which may positively regulate cell migration, is ER-dependent in ER-positive breast cancer cells. (PMID: 29180066)
- This study observed a progressive increase in Cx43 expression in the SOD1(G93A) mouse model of ALS during the disease course. Notably, this increase in Cx43 was also detected in the motor cortex and spinal cord of ALS patients. (PMID: 27083773)
- Data show that lymph node metastatic adenoid cystic carcinoma cells (AdCC) acquire cancer stem cell features involving the up-regulation of nicotinamide N-Methyltransferase and the loss of gap junction protein alpha-1, leading to epithelial-mesenchymal transition and consequent AdCC metastasis. (PMID: 29277772)
- Data show that Cx43 was inhibited predominantly via IL-1beta-activated ERK1/2 and p38 MAP kinase cascades. (PMID: 28938400)
- BMP2 decreases gap junction intercellular communication of luteinized human granulosa cells by downregulating Cx43 expression through an ALK2/ALK3-mediated SMAD-dependent signaling pathway. (PMID: 27986931)
- Nitric oxide (NO) controls calcium signal propagation through Cx37-containing gap junctions. The tyrosine phosphatase SHP-2 is the essential mediator and NO target. (PMID: 29025706)
Q&A
What criteria should I use when selecting a GJA1 antibody for my research?
When selecting a GJA1 antibody, consider these critical factors:
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Target epitope location: Antibodies targeting different regions of GJA1 may yield different results. For instance, C-terminal antibodies can detect both full-length GJA1-43k and the truncated GJA1-20k isoform .
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Validated applications: Verify the antibody has been validated for your specific application (WB, IHC-P, IF, ICC) with published validation images .
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Species reactivity: Confirm cross-reactivity with your experimental species. While many GJA1 antibodies work with human, mouse, and rat samples, specific validation is important for other species .
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Formulation compatibility: Consider whether the antibody formulation (containing BSA, sodium azide, etc.) is compatible with your downstream applications .
Always review validation images provided by manufacturers to assess specificity, background levels, and expected localization patterns before making your selection.
How can I validate the specificity of my GJA1 antibody?
To validate GJA1 antibody specificity:
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Positive control selection: Use tissues known to express high levels of GJA1 such as heart muscle, brain, or pigmented layer of retina .
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Knockdown validation: Compare staining in wild-type versus GJA1 siRNA-treated or CRISPR/Cas9 knockout cells to confirm specificity .
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Blocking peptide assay: Perform parallel experiments with and without a GJA1 blocking peptide to confirm signal specificity .
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Multiple antibody comparison: Use antibodies from different sources targeting different epitopes to confirm consistent localization patterns.
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Expected molecular weight verification: Confirm the detection of expected ~43 kDa band for full-length GJA1 in Western blot applications .
For quantitative applications, consider including validation of antibody linearity across a range of protein concentrations.
How should I optimize immunostaining protocols to detect different subcellular pools of GJA1?
GJA1 localizes to multiple subcellular compartments including gap junctions, cytoplasm, Golgi apparatus, and pericentriolar regions. Optimizing detection requires:
Protocol modifications by subcellular compartment:
For quantitative analysis of GJA1 distribution between compartments, consider line-scan analysis across cell borders or solubility fractionation followed by Western blot .
What approaches can differentiate between full-length GJA1-43k and the truncated GJA1-20k isoform?
Distinguishing between GJA1 isoforms requires specialized approaches:
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Antibody selection: Use antibodies targeting the C-terminus that can detect both GJA1-43k and GJA1-20k .
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Gel electrophoresis conditions: Employ 5-20% gradient SDS-PAGE gels running at 90V for optimal separation of different isoforms .
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Quantitative analysis: Calculate the ratio of GJA1-20k to GJA1-43k to assess changes in translation initiation .
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Genetic models: Consider using GJA1 M213L/M213L mouse models which have reduced GJA1-20k expression for functional studies .
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Fractionation approaches: Apply Triton X-100 solubility assays to separate junctional (insoluble) from non-junctional (soluble) GJA1 pools before Western blot analysis .
For accurate quantification, include both positive controls (cells overexpressing specific isoforms) and loading controls.
What are the optimal conditions for detecting GJA1 in cardiac tissue?
Cardiac tissue requires specific considerations for optimal GJA1 detection:
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Fixation: For paraffin-embedded sections, use 10% neutral-buffered formalin with careful processing to preserve gap junctions .
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Antigen retrieval: Perform heat-mediated antigen retrieval in citrate buffer (pH 6.0) for 20 minutes to maximize epitope accessibility .
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Antibody concentration: For immunohistochemistry, use 1:200 dilution (approximately 1-5 μg/ml) with 30-minute incubation at room temperature .
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Detection systems: For chromogenic detection, use HRP-conjugated secondary antibodies with DAB visualization; for fluorescence, use DyLight or Alexa Fluor conjugates .
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Blocking: Employ 10% goat serum blocking to minimize background .
For functional studies, correlate immunostaining with ECG assessments, as GJA1 deficiency can dramatically reduce R wave amplitude (by up to 92% in homozygous GJA1 M213L/M213L mice) .
How should I approach GJA1 detection in neuronal and retinal tissues?
For neuronal and retinal tissues:
Brain tissue preparation:
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Use fresh-frozen or carefully fixed tissue to preserve GJA1 structure
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Expect potential cross-reactivity with other connexin family members, requiring careful antibody selection
Retinal tissue considerations:
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For pigmented layer of retina, which highly expresses GJA1, optimize fixation to preserve both structure and antigenicity
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For frozen sections, prepare 10-μm thickness and mount on positively charged slides
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Include positive controls from heart tissue when validating new antibodies in retinal applications
When examining GJA1 in cilia formation studies, co-stain with ciliary markers such as acetylated tubulin and consider advanced imaging techniques such as structured illumination microscopy (SIM) for high-resolution analysis of Rab11-positive vesicles and GJA1 co-localization .
How can I address weak or absent GJA1 signal in Western blot applications?
When encountering weak or absent GJA1 signal in Western blots:
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Sample preparation: Optimize lysis conditions using buffers containing 1% SDS or RIPA buffer with protease inhibitors to effectively solubilize membrane proteins.
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Protein loading: Increase loading to 35-50 μg of total protein per lane as used in validated protocols .
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Transfer parameters: Use semi-dry transfer at 150mA for 50-90 minutes or wet transfer overnight at 30V/4°C for efficient transfer of membrane proteins .
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Antibody concentration: Increase primary antibody concentration to 0.5-1 μg/ml with overnight incubation at 4°C .
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Detection sensitivity: Employ enhanced chemiluminescent detection systems with extended exposure times (30 seconds to 5 minutes) .
If problems persist, consider tissue-specific optimization: heart tissue typically shows stronger signals than brain tissue, with consistent bands at approximately 43-45 kDa .
What strategies can address high background or non-specific binding in immunofluorescence applications?
For reducing background and improving specificity:
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Blocking optimization: Extend blocking time to 1.5 hours using 5-10% serum matched to secondary antibody species .
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Antibody titration: Perform serial dilutions (1:100 to 1:1000) to determine optimal antibody concentration.
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Secondary antibody controls: Include controls omitting primary antibody to assess secondary antibody specificity.
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Cross-adsorption: Use pre-adsorbed secondary antibodies to minimize cross-reactivity with endogenous immunoglobulins .
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Autofluorescence reduction: Apply Sudan Black B (0.1% in 70% ethanol) post-immunostaining to quench autofluorescence, particularly in tissues like heart with high autofluorescence.
For co-localization studies, carefully select compatible fluorophores with minimal spectral overlap and include single-label controls to confirm separation of signals.
How can I effectively study GJA1's role in ciliogenesis using antibody-based approaches?
Recent research has revealed GJA1's critical function in cilia formation and maintenance. To investigate this role:
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Experimental models: Use both primary cilium models (serum-starved RPE1 cells) and motile cilia models (Xenopus laevis embryonic multiciliated cells) .
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Knockdown approaches:
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Dominant-negative strategies: Express mutant forms of GJA1 (T154A, Δ130-136, or Δ234-243) to disrupt specific functions while maintaining protein expression .
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Co-localization analysis: Examine GJA1 distribution relative to:
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Functional readouts: Quantify cilia length, number, and beat frequency in control versus GJA1-depleted conditions .
For high-resolution analysis of subcellular distributions, employ structured illumination microscopy to visualize Rab11-positive vesicles and their relationship to GJA1 .
What approaches can assess the relationship between GJA1 and its binding partners?
To investigate GJA1 protein interactions:
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Co-immunoprecipitation: Use anti-GJA1 antibody-conjugated paramagnetic beads to pull down GJA1 and associated proteins .
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Sample preparation:
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Mass spectrometry analysis: Separate eluted proteins on SDS-PAGE gels, perform in-gel digestion, and analyze with high-resolution mass spectrometry (e.g., Orbitrap Fusion Lumos) .
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Validation approaches: Confirm interactions using reciprocal co-immunoprecipitation and Western blotting with specific antibodies against identified partners .
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Functional validation: Express dominant-negative GJA1 mutants and assess their ability to interact with binding partners compared to wild-type GJA1 .
This approach has successfully identified Rab11 and Rab8a as GJA1-binding partners involved in ciliogenesis regulation, demonstrating how antibody-based approaches can reveal novel protein functions .
