ARID3A Antibody
Description
Definition and Biological Context
ARID3A (AT-Rich Interaction Domain 3A), also known as Bright or DRIL1, is a DNA-binding protein critical for transcriptional regulation. It plays essential roles in:
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B cell development: Facilitating transitions during B cell maturation and immunoglobulin heavy chain (IgH) transcription .
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Hematopoietic lineage decisions: Regulating myeloid/erythroid differentiation and stem cell maintenance .
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Oncogenic processes: Collaborating with factors like E2F1 and RAS to influence cell cycle progression and cancer stemness .
The ARID3A antibody enables detection and quantification of this protein in experimental settings, supporting investigations into its mechanistic roles.
Hematopoietic Differentiation
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Inhibition of ARID3A in cord blood stem cells reduced B lineage cells and increased myeloid markers .
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Overexpression blocked myeloid/erythroid differentiation, highlighting its regulatory role in hematopoiesis .
E2F-Dependent Transcription
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ARID3A binds E2F target genes (e.g., Cdc2, cyclin E1) to modulate cell cycle progression .
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Chromatin immunoprecipitation (ChIP) assays confirmed ARID3A’s interaction with promoters of cyclin E1 and p107, influencing transcriptional activity .
Cancer and Stemness
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ARID3A collaborates with oncogenic RAS to immortalize fibroblasts and drive malignant transformation .
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ARID3B (a homolog) promotes cancer stemness in ovarian and squamous cell carcinomas, suggesting overlapping roles with ARID3A .
Technical Considerations
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Specificity: Antibodies like ab227274 show nuclear localization in HeLa cells and validate via IP-WB in HEK-293T lysates .
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Limitations: Cross-reactivity with ARID3B may occur due to structural similarities in their DNA-binding domains .
Future Directions
Research using ARID3A antibodies could explore:
Product Specs
Target Background
- Data indicate ARID3a(+) B cells as a type of effector B cell and link ARID3a expression in B lymphocytes to interferon alpha (IFNa)-associated inflammatory responses in systemic lupus erythematosus (SLE). PMID: 27522115
- Results suggest that proper regulation of ARID3a is crucial for the normal development of both myeloid and B lineage pathways. PMID: 26685208
- These data reveal novel functions for ARID3a in early hematopoiesis and suggest that understanding ARID3a levels in hematopoietic stem and progenitor cells (HSPCs) could be valuable for applications involving transplantation of these cells. PMID: 25535283
- Systemic lupus erythematosus (SLE) patients exhibited increased ARID3a+ B cells compared to healthy controls. ARID3a was not expressed in naive B cells of controls but was abundant in SLE patients. The number of ARID3a+ B cells correlated with disease activity. PMID: 25185498
- miR-125b can function as an oncogene in B-cell acute lymphoblastic leukemia by targeting ARID3a and mediating its repression. PMID: 22469780
- These findings support the hypothesis that Epstein-Barr virus EBNA1 initiates transcription at the C promoter through interactions between multiple EBNA1 homodimers and cellular transcription factors such as E2F1, ARID3A, and Oct-2. PMID: 22302879
- These results indicate both cooperative and interdependent roles for ARID3A and p53 in the transcriptional activation of p21(WAF1) in response to DNA damage. PMID: 22172947
- ARID3A functions as a critical antagonist to the p16(INK4A)-Rb tumor suppressor machinery by regulating promyelocytic leukemia protein stability. PMID: 22010578
- A study reports that E2FBP1 inhibits the accumulation of ICP0 RNA and, simultaneously, is degraded via ICP0's herpes ubiquitin ligase 2 (HUL-2) activity upon HSV-1 infection. PMID: 21248039
- Bright/ARID3a inhibition results in increased developmental plasticity in mouse and human cells. PMID: 20680960
- Solution NMR structure of the ARID domain of human ARID3A. PMID: 20455271
- Results demonstrate that DRIL1 disrupts cellular protection against RAS(V12)-induced proliferation downstream of the p19(ARF)/p53 pathway. PMID: 11812999
- E2FBP1 plays a role in the p53 regulatory pathway. PMID: 12136662
- Variations in Bright binding and matrix attachment region activity contribute to localized control of accessibility and, consequently, nonrandom gene use during V(D)J recombination. PMID: 12193717
- A putative p53-binding site was identified, which specifically responded to p53, in the second intron of the E2FBP1/DRIL1 gene. PMID: 12692263
- E2FBP1 modulates cell growth through down-regulation of promyelocytic leukemia bodies. PMID: 15017387
- Bright is not expressed in all human B lymphocyte subpopulations. PMID: 15203319
- TFII-I directly interacts with Bright through amino acids in Bright's protein interaction domain. PMID: 16738337
- Studies have identified Bright as a contributor to the accessibility of the IgH enhancer. PMID: 17386101
- Id1 inhibited DNA binding by Dril1, and the two proteins co-localized in vitro and in vivo, providing a potential mechanism for suppression of fibrosis by Id1 through inhibition of the profibrotic function of Dril1. PMID: 18583319
- A palmitoylated pool of Bright is diverted to lipid rafts of resting B cells where it associates with signalosome components. PMID: 19214191
Q&A
What experimental validations are necessary to confirm ARID3A antibody specificity in multi-application workflows?
Antibody validation requires orthogonal methods to confirm target engagement across applications. For ARID3A, which exhibits nuclear localization and variable expression in B cell subsets , researchers should:
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Western Blot (WB): Verify a single band at ~63–75 kDa in lysates from ARID3A-expressing cell lines (e.g., K-562) . Discrepancies between calculated (63 kDa) and observed (75 kDa) molecular weights may arise from post-translational modifications .
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Immunohistochemistry (IHC): Optimize antigen retrieval using TE buffer (pH 9.0) or citrate buffer (pH 6.0) to enhance epitope accessibility in formalin-fixed tissues .
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Flow Cytometry: Combine surface B cell markers (CD19, CD27, IgD) with intracellular ARID3A staining, using fluorescence-minus-one (FMO) controls to gate ARID3A+ populations accurately .
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Immunoprecipitation (IP): Pre-clear lysates with protein A/G beads to reduce non-specific binding, particularly in samples with low B cell counts .
Table 1: Validation Metrics for ARID3A Antibodies
| Application | Critical Validation Step | Common Pitfalls |
|---|---|---|
| WB | Knockout cell line comparison | Cross-reactivity with ARID3B |
| IHC | Tissue microarray staining concordance | Overfixation-induced epitope masking |
| Flow Cytometry | FMO controls for CD19+ subsets | False positives in activated B cells |
How do ARID3A+ B cell dynamics correlate with SLE disease activity in longitudinal studies?
In SLE cohorts, ARID3A+ B cells show temporal fluctuations linked to disease flares :
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Baseline Analysis: 43% of SLE patients (50/115) exhibit ARID3A+ B cell counts >2 SD above healthy controls at initial assessment .
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Longitudinal Tracking: 10/15 patients with high baseline ARID3A (ARID3A H) revert to normal levels (ARID3A N) during remission, while 8/22 ARID3A N patients develop transient ARID3A H states during flares .
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Statistical Modeling: Adjusted for total B cell counts, each log-unit increase in ARID3A+ naïve B cells correlates with a 1.4-point SLEDAI score rise (p<0.05) .
Methodological Considerations:
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Cohort Stratification: Define ARID3A H/N thresholds using healthy control means (e.g., >2 SD for H) .
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Confounding Factors: No correlations found with age, race, or immunosuppressants, necessitating mixed-effects models to account for inter-patient variability .
What technical challenges arise when quantifying ARID3A in memory B cell subsets?
Memory B cells (CD27+IgD−) pose unique challenges due to ARID3A’s bimodal expression:
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Subset Heterogeneity: Flow cytometry reveals two ARID3A expression peaks in SLE patients—one ARID3A− and one ARID3A+—requiring high-resolution gating .
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Low Abundance: ARID3A+ double-negative (DN) B cells (CD27−IgD−) constitute <5% of total B cells in controls but expand to >15% in ARID3A H patients .
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Fixation Artifacts: Prolonged formaldehyde fixation reduces ARID3A epitope detectability; limit fixation to ≤20 minutes for intracellular staining .
How can researchers resolve contradictions in ARID3A’s role in autoantibody production?
Despite correlations with total IgG/IgM levels, ARID3A+ B cell numbers do not predict specific autoantibody profiles . To address this paradox:
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Functional Assays: Perform B cell receptor (BCR) sequencing on sorted ARID3A+ vs. ARID3A− subsets to identify clonal expansion biases.
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Epigenetic Profiling: Use ChIP-seq to map ARID3A binding near immunoglobulin heavy chain (IgH) loci, which may regulate non-pathogenic vs. autoreactive antibody production .
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Cytokine Modulation: Co-culture ARID3A+ B cells with SLE-derived plasma cytokines (e.g., IFN-α) to test bystander activation hypotheses .
What advanced techniques enhance ARID3A detection in low-abundance samples?
For lymphopenic SLE samples or small tissue biopsies:
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Signal Amplification: Tyramide-based amplification in IHC improves detection in ARID3A-low plasmablasts .
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Spectral Flow Cytometry: 40-parameter panels enable simultaneous ARID3A quantification with 30+ B cell subset markers, reducing sample volume requirements .
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Digital Droplet PCR: Quantify ARID3A mRNA from CD19+ sorted cells to corroborate protein-level data .
How does ARID3A’s structural biology impact antibody development?
The ARID3A DNA-binding domain (residues 50–180) contains conserved AT-hook motifs that may sterically hinder antibody access. Successful epitope mapping strategies include:
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Recombinant Fragments: Express truncated ARID3A (e.g., N-terminal vs. C-terminal) to identify antibody-binding regions .
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Phage Display: Screen synthetic scFv libraries against the ARID domain to generate conformation-specific monoclonal antibodies .
What controls are essential when comparing ARID3A expression across autoimmune diseases?
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RA Controls: ARID3A+ B cell counts in rheumatoid arthritis (RA) patients mirror healthy donors, unlike SLE .
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Cohort Matching: Age/sex-match SLE, RA, and healthy cohorts to eliminate demographic confounders .
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Activation Markers: Include CD86 and CD43 staining to distinguish ARID3A’s role in activation vs. differentiation .
