Phospho-NFKBIA (Tyr305) Antibody
Description
Biological Context of NFKBIA and Tyr305 Phosphorylation
NFKBIA (Nuclear Factor Kappa B Inhibitor Alpha) is a key regulatory protein that binds to NF-κB transcription factors, sequestering them in the cytoplasm under basal conditions. Upon cellular stimulation (e.g., cytokines, pathogens, or stress), IκBα undergoes phosphorylation and degradation, enabling NF-κB to translocate to the nucleus and activate target genes .
Phosphorylation at Tyr305 is a post-translational modification implicated in:
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Signal-specific regulation: Unlike canonical serine phosphorylation (e.g., Ser32/36), Tyr305 phosphorylation occurs in response to distinct stimuli, such as oxidative stress or pervanadate treatment .
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Ubiquitination and degradation: Phosphorylated IκBα is recognized by E3 ubiquitin ligases, leading to proteasomal degradation and NF-κB activation .
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Modulation of protein interactions: Alters binding affinity for NF-κB subunits or other regulatory proteins .
Mechanistic Insights
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Western Blot Validation: The antibody detects phosphorylated IκBα in A431 (epidermoid carcinoma) and Jurkat (T-cell leukemia) cells treated with pervanadate, a phosphatase inhibitor .
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Functional Studies: Tyr305 phosphorylation modulates IκBα stability and NF-κB activity in pathways involving MAPK/ERK and DNA damage responses .
Disease Relevance
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Cancer: Dysregulated NF-κB signaling is common in malignancies. Tyr305 phosphorylation may influence tumor progression via altered inflammatory gene expression .
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Autoimmunity: Aberrant IκBα phosphorylation contributes to chronic inflammation .
Table 2: Select Vendors and Product Details
Technical Considerations
Product Specs
Target Background
- This study demonstrates changes in NF-kB expression levels induced by cell-free DNA in various cell types. PMID: 29743966
- Real-time PCR and western blotting revealed that Huaier extract decreased p65 and c-Met expression and increased IkappaBalpha expression, while paclitaxel increased p65 expression and reduced IkappaBalpha and c-Met expression. The molecular mechanisms may involve the inhibition of the NF-kappaB pathway and c-Met expression. PMID: 29039556
- Colorectal cancer cases showed an increased frequency of the NFkappaBIA-881G allele in Egyptian subjects. PMID: 28389768
- This study investigates the association between polymorphisms and the progression of chronic hepatitis B Virus infection among the Chinese Han population. PMID: 29093318
- miR-668 was upregulated in radioresistant human breast cancer cell lines MCF-7R and T-47DR, which targeted IkappaBalpha, activated the NF-kappaB pathway, and thus increased the radioresistance of breast cancer cells. PMID: 28138801
- This study shows that pristimerin suppressed tumor necrosis factor a (TNFalpha)-induced IkappaBa phosphorylation, translocation of p65, and expression of NFkappaB-dependent genes. Furthermore, pristimerin decreased cell viability and clonogenic ability of Uveal melanoma (UM) cells. A synergistic effect was observed in the treatment of pristimerin combined with vinblastine, a frontline therapeutic agent, in UM. PMID: 28766683
- These findings collectively demonstrate the functional importance of IkappaBalpha-mediated stripping of NFkappaB from DNA in the kinetic control of NFkappaB signaling. PMID: 28167786
- This study suggests that genetic polymorphisms of NFKB1A rs696, pre-miR-146a rs2910164, and pre-miR-499 rs3746444 may represent novel markers of AT susceptibility. PMID: 28674224
- XPO1 inhibitor combination therapy with bortezomib or carfilzomib induces nuclear localization of IkappaBalpha and overcomes acquired proteasome inhibitor resistance in human multiple myeloma. PMID: 27806331
- Molecular docking analysis indicated that transcription factor NF-kappaB was one of the potential molecular targets modulated by DTTF. Specifically, the drug blocked the TNFalpha-induced phosphorylation of upstream IkappaBalpha kinase in a time-dependent manner leading to the suppression of NF-kappaB activation and nuclear translocation. PMID: 27882436
- This study shows that HOTAIR regulates activation of NF-kB by decreasing Ik-Ba (NF-kB inhibitor) and establishes that by inducing prolonged NF-kB activation and expression of NF-kB target genes during DNA damage, HOTAIR plays a crucial role in cellular senescence and platinum sensitivity. PMID: 27041570
- This study reports amide hydrogen/deuterium exchange data revealing long-range allosteric changes in the NFkappaB (RelA-p50) heterodimer induced by DNA or IkappaBalpha binding. PMID: 28249778
- Sam68 is essential for DNA damage-induced NF-kappaB activation and colon tumorigenesis. PMID: 27458801
- Specifically, BCA2 serves as an E3 SUMO ligase in the SUMOylation of IkappaBalpha, which in turn enhances the sequestration of NF-kappaB components in the cytoplasm. Since HIV-1 utilizes NF-kappaB to promote proviral transcription, the BCA2-mediated inhibition of NF-kappaB significantly decreases the transcriptional activity of HIV-1. PMID: 28122985
- This study found that NFKBIA mRNAs were significantly expressed in normal tissues compared to glioma specimens. PMID: 27538656
- These findings indicate the prognostic value of NFKB inhibitor alpha (NFKBIA) in lower-grade gliomas (LGGs). PMID: 27052952
- W346 effectively inhibited tumor necrosis factor (TNF-a)-induced NF-kappaB activation by suppressing IKK phosphorylation, inhibiting IkB-a degradation, and restraining the accumulation of NF-kappaB subunit p65 nuclear translocation. W346 also affected NF-kappaB-regulated downstream products involved in cycle arrest and apoptosis. PMID: 26520440
- Treating cells with SZC014 resulted in a decrease in phosphorylation of IkBa and NF-kappaB/p65, as well as NF-kappaB/p65 nuclear translocation. The cytotoxic activities of seven OA derivatives were generally stronger than that of OA. Among these, SZC014 possessed the most potent anticancer activity in SGC7901 cells and could be a promising chemotherapeutic agent for the treatment of gastric cancer. PMID: 26547583
- Network analysis identified NFKBIA as a pathogenic gene in childhood asthma. PMID: 27420950
- HMBA was able to increase prostratin-induced phosphorylation and degradation of NF-kappaB inhibitor IkappaBalpha, thereby enhancing and prolonging prostratin-induced nuclear translocation of NF-kappaB, a prerequisite for stimulation of transcription initiation. PMID: 27529070
- Enhanced miR-381a-3p expression contributed to the injury of osteoarthritis mainly by inhibiting the expression of IkappaBalpha. PMID: 27312547
- Timely and efficient degradation of ubiquitinated IkappaB[alpha], concomitant with timely and efficient liberation of RelA from ubiquitinated IkappaB[alpha] and RelA nuclear translocation, essentially depends on the presence of functional p97/VCP. PMID: 26463447
- Activated Rac1 regulates the degradation of IkappaBalpha and the nuclear translocation of STAT3-NFkappaB complexes in starved cancer cells. PMID: 27151455
- This study reports a mutation in a Chinese patient that results in mycobacterial infections without ectodermal dysplasia. PMID: 26691317
- DAT stabilized IkBa by inhibiting the phosphorylation of Ika by the IkB kinase (IKK) complex. DAT induced proteasomal degradation of TRAF6, and DAT suppressed IKKb-phosphorylation through downregulation of TRAF6. PMID: 26647777
- This meta-analysis suggests that the rs3138053 polymorphism of NFKBIA gene is a candidate for susceptibility to overall cancers, while rs696 plays a protective role. PMID: 26488500
- This study identifies a novel BCR-ABL/IkappaBalpha/p53 network, whereby BCR-ABL functionally inactivates a key tumor suppressor in chronic myeloid leukemia. PMID: 26295305
- This study demonstrates an association between functional polymorphisms of IkappaBalpha rs696 and smoking with the risk of defective spermatogenesis, suggesting some interaction between the NF-kappaB signaling pathway and smoking-related ROS in human spermatogenesis. PMID: 25352423
- This study identifies a genetic variation associated with susceptibility to acute kidney injury. PMID: 26477820
- MicroRNA-19a mediates gastric carcinoma cell proliferation through the activation of IkappaBalpha. PMID: 26239140
- No association was observed between NFKBIA variants and risk of liver cancer. PMID: 24578542
- SM22alpha is a phosphorylation-regulated suppressor of IKK-IkappaBalpha-NF-kappaB signaling cascades. PMID: 25937534
- Data suggest that activity of IKBalpha can be regulated by dietary factors; dietary supplementation with luteolin inhibits vascular endothelial inflammation by suppressing IKBalpha/NFkappaB signaling. PMID: 25577468
- This review and meta-analysis of the association of NFKBIA -881 A>G polymorphism with cancer susceptibility reveals that -881 A>G polymorphism may increase the risk of cancer development in Asian populations. PMID: 26252270
- miR-126 may play a significant role in hepatic fibrosis by downregulating the expression of IkappaBalpha partly through the NF-kappaB signaling pathway. PMID: 25974152
- IkappaBetaalpha inhibits apoptosis at the outer mitochondrial membrane independently of NF-kappaB retention. PMID: 25361605
- The single nucleotide polymorphism rs1957106 CT and TT genotypes were found to be associated with lower NFKBIA protein levels and a poor prognosis of patients with glioblastoma. PMID: 25215581
- Data suggest that the NFKBIA 126 G/A polymorphism might be potentially helpful to identify liver transplant recipients with an increased susceptibility to develop recurrent acute rejections. PMID: 25112903
- Expression of IkappaBalpha in human bladder cancer cells is negatively correlated with epithelial-mesenchymal transition and tumor invasion in vitro. PMID: 25374080
- NFKBIA-rs2233419AG was associated with a significantly increased risk of developing recurrent wheezing. PMID: 25326706
- miR-196a can directly interact with IkappaBalpha 3'-UTR to suppress IkappaBalpha expression and subsequently promote activation of NF-kappaB. PMID: 24463357
- MiR-196a promotes pancreatic cancer progression by targeting nuclear factor kappa-B-inhibitor alpha. PMID: 24504166
- Data indicate that following bortezomib treatment, there was accumulation of IkappaB-alpha (IkappaBalpha) without affecting its phosphorylation status at an early time point. PMID: 23697845
- This study reveals that polymorphisms in the IkB-alpha promoter (-881 A/G, -826 C/T) are strongly associated with the susceptibility of Iranian Multiple Sclerosis patients. PMID: 24368589
- The results of this study suggest that oligodendroglial IkappaBalpha expression and NF-kappaB are activated early in the course of MSA, and their balance contributes to the decision of cellular demise. PMID: 24361600
- No statistically significant CRC risk association was found for the NFKBIA polymorphisms. PMID: 23996241
- Data indicate that NFKBIA deletions are present but not frequent in Glioblastoma multiforme (GBM). The deletions become frequent in GBM neurospheres (NS), an event that seems to be affected by the presence of EGF in the culture medium. PMID: 24330732
- The analysis of IkappaBalpha expression at salivary gland epithelial cell level could be a potential new hallmark of Sjogren's syndrome progression. PMID: 23377923
- IkappaBalpha sequesters not only p65 but also RPS3 in the cytoplasm. PMID: 24457201
- NF-kB expression was downregulated, and its cytoplasmic inhibitor IKBalpha was increased in CTLA4-Ig treated macrophages. PMID: 24295474
Q&A
What is Phospho-NFKBIA (Tyr305) Antibody and what cellular target does it specifically recognize?
Phospho-NFKBIA (Tyr305) Antibody specifically recognizes IκB-alpha (NFKBIA) when phosphorylated at the tyrosine 305 residue. This antibody is typically generated using a synthetic peptide corresponding to amino acid residues around tyrosine 305 of human IκB-alpha, with the peptide sequence having low homology to other IκB proteins .
The antibody detects a 38 kDa protein on SDS-PAGE immunoblots of A431 and Jurkat cells treated with pervanadate, but does not detect this band in control cells, confirming its specificity for the phosphorylated form . Most commercially available versions are polyclonal rabbit antibodies that have been cross-adsorbed to phospho-tyrosine coupled to agarose and then affinity purified using phospho-IκB-alpha (Tyr-305) peptide without carrier to ensure specificity .
What is the biological significance of NFKBIA and its phosphorylation at Tyr305?
NFKBIA (IκB-alpha) serves as a critical regulator of the NF-κB signaling pathway by inhibiting the activity of dimeric NF-kappa-B/REL complexes. It functions by trapping REL dimers (specifically RELA/p65 and NFKB1/p50) in the cytoplasm through masking their nuclear localization signals .
The phosphorylation at Tyr305, located in the C-terminal PEST domain of NFKBIA, represents a distinct regulatory mechanism from the classical IKK-mediated phosphorylation at Ser32/36. In B cells stimulated with anti-IgM, Bruton's tyrosine kinase (Btk) is rapidly activated and phosphorylates IκBα at Tyr305 and Tyr289 residues . This phosphorylation contributes to NFKBIA degradation and subsequent NF-κB activation, allowing NF-κB dimers to translocate to the nucleus and initiate gene transcription .
What experimental applications are suitable for Phospho-NFKBIA (Tyr305) Antibody?
Phospho-NFKBIA (Tyr305) Antibody can be utilized in multiple experimental applications:
Western blotting represents the most thoroughly validated application, with specific validation in A431 and Jurkat cell models treated with pervanadate .
What are the optimal storage conditions for maintaining Phospho-NFKBIA (Tyr305) Antibody activity?
For optimal antibody stability and performance:
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Can be refrigerated at 2-8°C for up to 2 weeks for ongoing experiments
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Typical formulation includes PBS with 50% glycerol, 0.5% BSA and 0.02% sodium azide as preservative
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To prevent activity loss from freeze-thaw cycles, store in small aliquots
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Most formulations report stability for at least 1 year at -20°C when properly stored
How does the phosphorylation of NFKBIA at Tyr305 differ functionally from other phosphorylation sites?
NFKBIA undergoes multiple phosphorylation events with distinct functional outcomes:
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Tyr305 phosphorylation: Located in the C-terminal PEST domain, this modification occurs in B cells stimulated with anti-IgM through Btk activation . Unlike Ser32/36 phosphorylation, Tyr305 phosphorylation represents an alternative pathway for NF-κB activation.
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Ser32/36 phosphorylation: The canonical pathway involves IKK complex activation by various stimuli (LPS, viral proteins, cytokines) leading to phosphorylation of Ser32/36 in the N-terminal SRD region . This triggers ubiquitination and proteasomal degradation.
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Tyr42 phosphorylation: According to GeneCards, "phosphorylation at Tyr-42 activates NF-kappa-B without triggering proteolytic degradation of NFKBIA" , suggesting a mechanism distinct from both Ser32/36 and Tyr305 pathways.
The PEST domain, where Tyr305 is located, contains multiple phosphorylation sites (Ser283, Ser289, Ser293, Thr291 and Thr299) , suggesting complex, potentially context-dependent regulation of NFKBIA function through this region.
What are the optimal experimental conditions for detecting NFKBIA Tyr305 phosphorylation?
For robust detection of Tyr305 phosphorylation:
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Cell stimulation protocols:
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Western blotting optimization:
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Validation controls shown in scientific data:
Western blot analysis should include:
What are effective validation strategies to confirm Phospho-NFKBIA (Tyr305) Antibody specificity?
Several validation approaches can confirm antibody specificity:
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Peptide competition assays:
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Kinase inhibition:
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Genetic approaches:
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NFKBIA knockout/knockdown cells as negative controls
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Cells expressing NFKBIA Y305F mutant (tyrosine to phenylalanine) to prevent phosphorylation
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Mass spectrometry:
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Confirm phosphorylation at Tyr305 by immunoprecipitation followed by MS analysis
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Map phosphorylation sites in response to different stimuli
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How can researchers quantify changes in NFKBIA Tyr305 phosphorylation across different experimental conditions?
For accurate quantification of phosphorylation dynamics:
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Western blot quantification:
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Always normalize phospho-NFKBIA signal to total NFKBIA levels
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Use infrared fluorescence-based systems for wider linear dynamic range
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Include multiple biological and technical replicates (minimum n=3)
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Employ time-course analysis to capture transient phosphorylation events
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ELISA-based approaches:
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Image analysis for immunofluorescence/IHC:
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Quantify nuclear vs. cytoplasmic distribution to correlate with functional outcomes
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Use automated image analysis software with consistent thresholding
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Phospho-proteomics:
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Targeted MS/MS approaches for absolute quantification
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SILAC or TMT labeling for relative quantification across conditions
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What is known about the three-dimensional structural changes in NFKBIA following Tyr305 phosphorylation?
The structural implications of Tyr305 phosphorylation must be understood in context of NFKBIA's domain architecture:
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NFKBIA structure:
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PEST domain characteristics:
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Functional consequences:
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The interaction between PEST region and p65 NTD involves acidic patches (Glu282, Glu284, Asp285, Glu286, Glu287) in PEST and basic regions in p65 NTD
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Phosphorylation at Tyr305 potentially disrupts this interaction, affecting the allosteric regulation of DNA binding by NF-κB
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May alter the "locked" conformation of the NTD of p65, allowing DNA binding
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Though direct structural data on Tyr305 phosphorylation is limited, understanding the role of PEST domain in NF-κB regulation provides insight into potential mechanisms.
How does cell type and stimulus specificity affect NFKBIA Tyr305 phosphorylation patterns?
NFKBIA Tyr305 phosphorylation exhibits context-dependent patterns:
Understanding these context-specific patterns is crucial for interpreting experimental results and designing appropriate positive controls when using Phospho-NFKBIA (Tyr305) Antibody.
