Phospho-GJA1 (S261) Antibody
Product Specs
Target Background
- The LB2003 cells, devoid of three key K(+) uptake transport mechanisms, cannot grow in low-[K(+)] medium, but expression of Cx26, Cx43, or Cx46 rescues their growth defect (growth complementation PMID: 27789753).
- A novel role for Cx43-formed unidirectional gap junctional intercellular communication was in mediating metabolic coupling between cancer-associated fibroblasts and non-small cell lung cancer cells and thereby facilitating malignant progression of NSCLC by enhancing oxidative phosphorylation and increasing ATP-activated PI3K/Akt and MAPK/ERK signaling pathways. PMID: 30453281
- Studies have demonstrated overexpression of Ubc9 protein in osteosarcoma. Silencing Ubc9 in osteosarcoma cell lines induced decoupling of SUMO1 from Cx43, generating increased free Cx43 levels, which is important for reconstructing gap junction intercellular communication and recovering cellular functions. PMID: 29956745
- These results demonstrate that the Cx43 SH3-binding domain, in addition to the CT9 region, critically controls hemichannel activity at high [Ca(2+)]i, which may be involved in pathological hemichannel opening. PMID: 29218600
- Pinocembrin alleviated ventricular arrhythmia in I/R rats via enhancing Na+-K+ATPase and Ca+-Mg2+ATPase activity and upregulating Cx43 and Kir2.1 protein expression. PMID: 30022020
- A Tunisian family with ODDD marked by neurologic signs with anticipation which is uncommon in this disease and extends the mutational spectrum of the GJA1 gene by 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) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating 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 labour, 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 labour 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
- Studies have found a significant difference in the expression of Cx43 and SUMO1 between cancer stem cells and non-cancer stem cells in liver cancer. By the co-expression of Cx43 and SUMO1 in cancer stem cells, the gap junction intercellular communication of liver cancer stem cells was obviously improved. 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 atrial septal defect. PMID: 29198211
- Inhibition of Connexin43 signalling 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 result 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 an important role in the formation of vascular intima thickening in patients with moyamoya disease. PMID: 29395647
- Findings demonstrate how SRC3 and Cx43 regulation between BMSCs and myeloma cells mediate cell growth and disease progression. PMID: 29075794
- We generated mutations of known conserved regulatory serine (S) residues 255, 279/282, 365, 368, and 373. 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. Our results suggest that 1) S365 and S373 phosphorylation promote forward trafficking, In addition, phosphorylation on these residues appears to prevent premature binding of ZO-1 PMID: 29021339
- These data suggest that chronic exposure to glucose-evoked TGFbeta1 induce 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, tunnelling nanotubes or gap junctions. (Review) PMID: 29025971
- One novel homozygous variant c.169C>T and one heterozygous SNP c.624C>T (rs530633057) were determined 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, which suggests a novel susceptibility gene for Chinese sudden unexplained nocturnal death syndrome. PMID: 27992820
- The functional modulation of connexin 43 (Cx43) indicate 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 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 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 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 the H9c2 cells against hyperglycemia-induced apoptosis and Cx43 down-regulation, in part, mediated through the induction of 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 that has independently antioncogenic 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
- MIF is involved in the pathogenesis of AF, 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
- We present 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
- The complex regulatory and signalling networks controlled by the Cx43 CT, including the extensive protein interactome that underlies both gap junction channel-dependent and -independent functions. PMID: 28526583
- Cx43 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 concentration 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
- Studies highlight 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 [Ca2+]i. 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
- We suggest 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
- NO controls the 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 is the significance of phosphorylation at S261 in Connexin 43 (GJA1)?
Phosphorylation at serine residue 261 (S261) in Connexin 43 (GJA1) plays a critical role in regulating gap junction dynamics and cellular communication. Connexin 43 is a key protein forming gap junctions, which are essential for intercellular communication in various tissues, including cardiac and neural tissues . Phosphorylation events at specific residues, such as S261, influence the assembly, gating, and degradation of gap junctions. Studies have shown that phosphorylation at S261 contributes to connexosome formation or processing, which are vesicles derived from internalized gap junction plaques .
Experimental evidence using immunogold electron microscopy has demonstrated that phosphorylation at S261 is selectively associated with connexosomes but not with intact gap junctions . This suggests that S261 phosphorylation may be involved in the turnover or internalization processes rather than the active communication functions of gap junctions.
How can researchers distinguish between phosphorylated and non-phosphorylated forms of Connexin 43 at S261?
To distinguish between phosphorylated and non-phosphorylated forms of Connexin 43 at S261, researchers typically employ antibodies that are specific to the phosphorylated state of the protein. The Phospho-GJA1 (S261) antibody detects endogenous levels of Connexin 43 only when phosphorylated at S261 . These antibodies are affinity-purified using epitope-specific immunogens derived from peptides surrounding the phosphorylation site.
Immunohistochemistry (IHC), immunofluorescence (IF), and enzyme-linked immunosorbent assay (ELISA) are commonly used techniques for this purpose . For example, IHC can be used to visualize the localization of phosphorylated Connexin 43 in tissue sections, while ELISA provides quantitative measurements of phosphorylation levels.
Additionally, double labeling techniques can be employed to confirm the presence of total Connexin 43 alongside its phosphorylated forms. This involves labeling adjacent sections with different antibodies targeting total and phosphorylated epitopes .
What experimental models are suitable for studying phosphorylation at S261?
Experimental models suitable for studying phosphorylation at S261 include both in vitro cell culture systems and in vivo animal models. Human embryonic stem cell-derived cardiomyocytes have been used to investigate the role of phosphorylation in cardiac tissues under ischemic conditions . These cells are cultured under controlled conditions and exposed to hypoxia or other stressors to mimic pathological states.
Transgenic mouse models offer another powerful tool for studying phosphorylation events. For instance, mice engineered with phosphomimetic mutations at specific residues of Connexin 43 have been used to study the effects of phosphorylation on gap junction remodeling and arrhythmia susceptibility . Such models provide insights into how phosphorylation influences physiological and pathological processes.
How does phosphorylation at S261 affect gap junction assembly and function?
Phosphorylation at S261 appears to primarily influence gap junction turnover rather than assembly or active communication functions. Studies using electron microscopy have shown that phosphorylated S261 is predominantly localized on connexosomes, which are vesicles formed during the internalization of gap junction plaques . This suggests that S261 phosphorylation may play a role in the disassembly or degradation processes.
In contrast, other phosphorylation sites such as S368 have been linked to reduced intercellular communication due to gap junction disassembly triggered by protein kinase C-mediated phosphorylation . Therefore, while S368 phosphorylation directly impacts gap junction activity, S261 phosphorylation seems more involved in post-assembly processes.
What methods can be used to quantify phosphorylation levels at S261?
Quantification of phosphorylation levels at S261 can be achieved using ELISA, Western blotting, or mass spectrometry-based approaches. ELISA is particularly useful for high-throughput analysis and provides quantitative data on antibody binding specific to phosphorylated S261 .
Western blotting allows researchers to separate proteins by molecular weight and detect phosphorylated forms using specific antibodies. The use of phospho-specific antibodies ensures that only proteins phosphorylated at S261 are detected.
Mass spectrometry offers an advanced method for quantifying phosphorylation levels with high sensitivity and specificity. It can identify multiple post-translational modifications simultaneously and provide detailed insights into the phosphorylation dynamics under different experimental conditions.
What challenges might arise when interpreting data from Phospho-GJA1 (S261) antibody experiments?
One major challenge in interpreting data from Phospho-GJA1 (S261) antibody experiments is ensuring specificity for the phosphorylated form of Connexin 43 at S261. Cross-reactivity with other phosphorylated residues or non-phosphorylated forms can lead to misleading results . Proper validation of antibody specificity through controls such as peptide competition assays is essential.
Another challenge involves distinguishing between functional effects of phosphorylation at different residues. For example, while S368 phosphorylation reduces intercellular communication by triggering gap junction disassembly, S261 phosphorylation appears more related to connexosome formation . Understanding these distinct roles requires careful experimental design and interpretation.
Finally, variations in experimental conditions such as antibody dilution, incubation times, and tissue preparation can affect reproducibility. Standardizing protocols across studies is crucial for obtaining reliable data.
How does ischemia influence phosphorylation patterns in Connexin 43?
Ischemia has been shown to significantly alter the phosphorylation patterns in Connexin 43, including its phosphorylated forms at S261. Acute ischemic stress promotes internalization of gap junctions by modifying their phosphorylation status . This leads to decreased sarcolemmal Connexin 43 levels and impaired electrical coupling between neighboring cardiomyocytes.
In transgenic mouse models with phosphatase-resistant mutations at CK1-targeted residues (e.g., S325/328/330), resistance to ischemia-induced remodeling has been observed . These findings suggest that specific phosphorylation events mediate protective or detrimental responses during ischemia.
Experimental protocols often involve exposing cells or tissues to hypoxic conditions followed by analysis using phospho-specific antibodies or imaging techniques .
What role does casein kinase 1 play in regulating Connexin 43 phosphorylation?
Casein kinase 1 (CK1) is one of several kinases known to phosphorylate Connexin 43 at multiple serine residues within its carboxy terminus . CK1-mediated phosphorylation modulates gap junction assembly and turnover. For example, CK1 targets residues such as S325/328/330, which influence gap junction remodeling during ischemia.
Phosphomimetic mutations introduced into these sites have demonstrated reduced susceptibility to arrhythmias and attenuated remodeling under pathological conditions . These findings highlight CK1's critical role in regulating Connexin 43 function through site-specific phosphorylation.
