ARID5 Antibody

What is ARID5B and what cellular functions make it a target for antibody development?

ARID5B (AT-Rich Interactive Domain 5B) is a transcription coactivator that binds to the 5'-AATA[CT]-3' core sequence in DNA and plays crucial roles in adipogenesis and liver development. It functions by forming a complex with phosphorylated PHF2, which mediates demethylation at specific lysine residues. This PHF2-ARID5B complex targets promoters where PHF2 demethylates dimethylated 'Lys-9' of histone H3 (H3K9me2), leading to transcriptional activation of target genes . The complex also acts as a coactivator of HNF4A in liver tissue. ARID5B is required for adipogenesis through its regulation of triglyceride metabolism in adipocytes by controlling expression of adipogenic genes . Recent research has also identified ARID5B as a negative modulator of IL-6 production in rheumatoid arthritis synovial fibroblasts, suggesting potential therapeutic applications in inflammatory conditions .

When designing experiments with ARID5B antibodies, researchers should consider its multiple cellular functions and tissue-specific expression patterns to appropriately interpret results.

What types of ARID5B antibodies are available for research applications?

Several ARID5B antibodies are available for research, varying in host species, clonality, targeted regions, and applications. The most common types include:

• Polyclonal antibodies: These are raised in rabbits against specific regions of ARID5B, such as the C-terminal region. For example, catalog ABIN2784295 is a rabbit polyclonal antibody targeting the C-terminal region of human ARID5B .

• Monoclonal antibodies: Some manufacturers offer mouse monoclonal antibodies against ARID5B, such as clone 1C2 targeting amino acids 1483-1582 .

• Region-specific antibodies: Different antibodies target specific amino acid regions of ARID5B:

  • C-terminal region (example: ABIN2784295)

  • AA 1031-1080 region

  • AA 389-418 region

  • AA 1483-1582 region

  • AA 77-126 region

• Host species: Most commonly rabbit for polyclonals and mouse for monoclonals .

• Conjugation status: Most are unconjugated, though some conjugated versions (e.g., with APC) exist for specialized applications .

When selecting an ARID5B antibody, researchers should consider the specific region they wish to target, especially when studying different isoforms, as both long and short isoforms of ARID5B have been identified with potentially different functional properties .

What are the validated applications for ARID5B antibodies?

ARID5B antibodies have been validated for several research applications, with varying degrees of optimization depending on the specific antibody:

• Western Blotting (WB): Most ARID5B antibodies are validated for Western blotting, including ABIN2784295 and ab226776 . This application allows detection of ARID5B protein expression levels and isoform analysis.

• Immunocytochemistry (ICC): Some antibodies are validated for cellular localization studies .

• Immunofluorescence (IF): Useful for visualizing ARID5B localization within cells and tissues .

• Immunohistochemistry on paraffin sections (IHC-P): Allows for detection of ARID5B in fixed tissue samples .

• ELISA: Select antibodies have been validated for ELISA applications, enabling quantitative measurement of ARID5B levels .

When planning experiments, researchers should verify the validation status for their specific application and consider performing preliminary validation in their experimental system, particularly when studying different cell types or when distinguishing between ARID5B isoforms, as both long and short isoforms have been identified .

How can I optimize Western blotting protocols specifically for ARID5B detection?

Optimizing Western blotting for ARID5B detection requires addressing several technical considerations:

• Sample preparation:

  • ARID5B is primarily a nuclear protein when unstimulated, but can shuttle upon lipopolysaccharide stimulation in immune cells . Consider using nuclear extraction protocols.

  • Include protease inhibitors to prevent degradation.

  • When studying both long and short isoforms, ensure your gel percentage can resolve these different molecular weight variants.

• Gel selection and separation:

  • Use 8-10% SDS-PAGE gels for optimal separation of ARID5B's high molecular weight.

  • Consider gradient gels (4-15%) if analyzing multiple isoforms simultaneously.

• Antibody selection and validation:

  • Use antibodies targeting conserved regions when working across species, as ARID5B shows high sequence homology: Cow (100%), Guinea Pig (86%), Horse (100%), Human (100%), Mouse (93%), Rabbit (100%), Rat (93%) .

  • For isoform-specific detection, select antibodies targeting unique regions of either the long or short isoform.

• Blocking and antibody incubation:

  • BSA-based blocking solutions may yield lower background than milk-based solutions.

  • Extended primary antibody incubation (overnight at 4°C) often improves specific signals.

• Positive controls:

  • Include cell lines known to express ARID5B (e.g., lymphoblastoid cell lines like GM12878 or Nalm6 for studies related to hematological contexts) .

• Anticipated results:

  • Be aware that TNF-α negatively regulates ARID5B expression in synovial fibroblasts, which may affect detection levels in inflammatory conditions .

  • Both long and short isoforms may be detected, with the long isoform having functional significance in IL-6 regulation .

This optimization approach enables more reliable detection of ARID5B in complex biological samples and accurate quantification of expression changes in experimental conditions.

What is the role of ARID5B in disease pathogenesis and how can antibodies facilitate this research?

ARID5B has been implicated in several disease processes, with antibodies serving as critical tools for understanding its pathogenic mechanisms:

• Acute Lymphoblastic Leukemia (ALL):

  • Targeted sequencing of ARID5B in germline DNA of 5008 children with ALL revealed variants associated with disease susceptibility .

  • ARID5B antibodies can be used in ChIP assays to identify altered binding patterns associated with risk variants.

  • Cis-regulatory elements in ARID5B have been systematically identified using dCas9-KRAB-mediated enhancer interference systems in ALL cells .

• Rheumatoid Arthritis (RA):

  • ARID5B functions as a negative modulator of IL-6 production in RA synovial fibroblasts .

  • The long isoform specifically suppresses TNF-α-stimulated IL-6 production .

  • The RA risk allele rs10821944 in ARID5B intron 4 shows eQTL effects on long isoform expression in TNF-α-treated synovial fibroblasts .

  • Antibodies distinguishing between isoforms can help determine the differential roles in inflammatory responses.

• Metabolic Disorders:

  • ARID5B regulates triglyceride metabolism in adipocytes and is required for adipogenesis .

  • Antibodies can track ARID5B expression changes during adipocyte differentiation and in metabolic disease models.

• Experimental Approaches:

  • siRNA screening combined with antibody detection has identified ARID5B among 7 transcription factors affecting IL-6 production .

  • Lentiviral overexpression models with subsequent antibody validation can distinguish functional effects of different ARID5B isoforms .

  • eQTL analysis utilizing antibodies can correlate genotype with protein expression levels to understand disease risk mechanisms .

By employing specific antibodies in these research contexts, investigators can better understand how ARID5B variants contribute to disease susceptibility and potentially identify new therapeutic targets.

How can I investigate the dual DNA/RNA binding capabilities of ARID5B using antibodies?

Investigating ARID5B's dual nucleic acid binding capabilities requires specialized experimental approaches:

• Chromatin Immunoprecipitation (ChIP):

  • Use anti-ARID5B antibodies to immunoprecipitate protein-DNA complexes.

  • Focus on AT-rich DNA regions, as ARID5B shows preference for the 5'-AATA[CT]-3' core sequence .

  • Analysis of pulled-down DNA sequences can identify genomic binding sites.

  • Recent research indicates that the ARID domain can distinguish between specific DNA and RNA-binding motifs .

• RNA Immunoprecipitation (RIP) and CLIP:

  • Employ anti-ARID5B antibodies for RNA immunoprecipitation to identify RNA targets.

  • Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP2) has been used to map ARID5B binding sites throughout the transcriptome .

  • RBNS (RNA Bind-n-Seq) has revealed a preference for CAGGCAG consensus motif, accompanied by a general preference for AU-rich motifs .

• Structural considerations:

  • The intrinsically disordered regions (IDRs) flanking the ARID domain modulate specificity and affinity of DNA binding .

  • These IDRs appear crucial for RNA interactions .

  • When selecting antibodies, consider those that will not interfere with these interactions.

• Functional validation:

  • In immune cells, ARID5B has been shown to counteract the degradation of specific mRNAs (like IL-6) mediated by regulatory RNA-binding proteins Regnase-1 and Roquin .

  • ARID5B stabilizes Ox40 mRNA by interacting with stem-loop structures in the 3'-UTR .

  • Antibody-based detection can track ARID5B shuttling between nucleus and cytoplasm upon stimulation, which is relevant to its RNA-protective functions .

• Methodological protocol:

  • For NMR spectroscopy observations of atom-resolved binding:

    • Core ARID domain (aa 49-152) shows marginal interactions with RNA .

    • Extended ARID domain with flanking regions shows stronger interactions.

    • This highlights the importance of selecting antibodies that recognize appropriate epitopes for studying these interactions.

This multifaceted approach enables comprehensive investigation of ARID5B's dual role in DNA and RNA binding, with antibodies serving as essential tools throughout the experimental workflow.

What are the functional differences between ARID5B isoforms and how can antibodies distinguish them?

ARID5B exists in multiple isoforms with distinct functional properties, and specialized antibody approaches are required to differentiate them:

• Isoform characteristics:

  • Both long and short isoforms of ARID5B are expressed in rheumatoid arthritis synovial fibroblasts (RASFs) .

  • Both isoforms are negatively regulated by TNF-α in RASFs .

  • The long isoform specifically suppresses IL-6 production stimulated with TNF-α, indicating functional divergence between isoforms .

• Antibody selection for isoform discrimination:

  • Select antibodies targeting unique epitopes present in one isoform but absent in others.

  • Antibodies targeting the C-terminal region (e.g., ABIN2784295) may recognize multiple isoforms .

  • Consider using antibodies targeting specific amino acid regions that differ between isoforms: AA 1031-1080, AA 389-418, AA 1483-1582, or AA 77-126 .

• Experimental validation approaches:

  • Western blotting with isoform-specific antibodies can quantify relative expression levels.

  • Immunoprecipitation followed by mass spectrometry can confirm antibody specificity for particular isoforms.

  • siRNA knockdown experiments targeting specific isoforms, followed by antibody detection, have revealed differential regulation of IL-6 production .

  • Lentiviral overexpression systems have been used to independently verify the functions of long and short isoforms .

• Genetic association with isoform expression:

  • eQTL analysis using 58 synovial fibroblasts demonstrated that the RA risk allele rs10821944 in ARID5B intron 4 affects expression of the long isoform specifically in TNF-α-treated cells .

  • This genetic-molecular correlation suggests isoform-specific disease relevance.

• Methodological considerations:

  • When designing experiments to distinguish isoforms, include positive controls expressing known isoforms.

  • Consider using recombinant isoforms as standards for antibody validation.

  • In functional studies, complement antibody-based detection with RT-qPCR using isoform-specific primers.

Understanding the functional differences between ARID5B isoforms has significant implications for disease mechanisms and potential therapeutic targeting, particularly in inflammatory conditions where isoform-specific functions have been identified.

How can CRISPR-based approaches complement antibody studies of ARID5B function?

CRISPR-based technologies offer powerful complementary approaches to antibody studies for investigating ARID5B function:

• Reporter systems for enhancer analysis:

  • CRISPR/Cas9 genome editing has been used to insert mCherry at the 3' end of the ARID5B coding frame in human ALL cell line Nalm6, creating a reporter system where mCherry expression directly reflects ARID5B transcription .

  • This system enables measurement of the effects of cis-regulatory elements on ARID5B expression.

• CRISPRi for enhancer mapping:

  • The CRISPRi system, in which transcription repressor KRAB is targeted to enhancers by sgRNAs, has been used to systematically test putative cis-regulatory elements affecting ARID5B expression .

  • This approach identified 6 statistically significant regulatory elements from 21 predicted enhancers .

  • Particularly, the intron 3 CRE (chr10: 61,957,118-61,967,424) showed the strongest effects on ARID5B expression .

• Validation workflow combining CRISPR and antibodies:

  • CRISPR-generated models with altered ARID5B expression can be validated using antibody-based detection methods.

  • Western blotting with anti-ARID5B antibodies can confirm knockout or altered expression.

  • Chromatin immunoprecipitation using ARID5B antibodies can verify altered DNA binding in CRISPR-modified models.

• Studying variant effects:

  • CRISPR-based homology-directed repair can introduce specific ARID5B variants associated with disease risk.

  • Antibody-based studies can then assess how these variants affect protein expression, localization, and function.

  • This approach has been valuable for studying how variants disrupt transcription factor binding .

• Experimental design considerations:

  • When combining CRISPR and antibody approaches, ensure antibody epitopes are not affected by CRISPR-introduced modifications.

  • Include appropriate controls to distinguish effects of CRISPR editing from potential off-target effects.

  • For enhancer studies, compare results from CRISPRi with traditional antibody-based ChIP-seq data to comprehensively map regulatory elements.

This integrated approach leveraging both CRISPR technology and antibody-based detection methods provides more robust insights into ARID5B function than either methodology alone, particularly for complex regulatory mechanisms and variant effects.

How can I validate the specificity of an ARID5B antibody for my particular experimental system?

Validating ARID5B antibody specificity is crucial for experimental reliability. Implementation of the following comprehensive validation protocol is recommended:

• Positive and negative controls:

  • Positive controls: Use cell lines known to express ARID5B, such as lymphoblastoid cell line GM12878 or Nalm6 cells .

  • Negative controls: Include knockout or knockdown samples (siRNA or CRISPR-based) to confirm signal loss.

  • Recombinant protein: Test antibody against recombinant ARID5B protein (full-length or fragments) if available.

• Cross-reactivity assessment:

  • Consider predicted reactivity across species: Cow (100%), Guinea Pig (86%), Horse (100%), Human (100%), Mouse (93%), Rabbit (100%), Rat (93%) .

  • When working with non-human samples, verify epitope conservation in your species of interest.

• Epitope mapping and isoform considerations:

  • Determine which ARID5B region your antibody targets (e.g., C-terminus, specific amino acid regions) .

  • For experiments involving multiple isoforms, ensure your antibody can detect the relevant isoform or distinguish between long and short variants .

• Application-specific validation:

  • Western blot: Confirm a single band at the expected molecular weight (~150 kDa for full-length protein, with variations for isoforms).

  • Immunoprecipitation: Verify enrichment by mass spectrometry or Western blot of the precipitate.

  • ChIP/RIP: Include IgG controls and known target regions as positive controls.

• Stimulation responses:

  • Test antibody detection under conditions known to alter ARID5B expression, such as TNF-α treatment, which negatively regulates ARID5B in synovial fibroblasts .

  • Verify nuclear/cytoplasmic shuttling in response to lipopolysaccharide stimulation in immune cells .

• Functional validation:

  • Correlate antibody-detected expression levels with functional readouts, such as IL-6 production in RASFs .

  • Verify ability to detect ARID5B in protein complexes (e.g., with phosphorylated PHF2) .

This systematic validation approach ensures reliable antibody performance in your specific experimental system, reducing the risk of false positives or negatives and increasing confidence in your research findings.

What experimental approaches can resolve contradictory findings about ARID5B function in different cell types?

Resolving contradictory findings about ARID5B function across different cellular contexts requires methodologically rigorous approaches:

• Context-specific expression profiling:

  • Quantify ARID5B isoform expression across cell types using validated antibodies and RT-qPCR.

  • In rheumatoid arthritis synovial fibroblasts, both long and short isoforms are expressed but have different functional impacts on IL-6 production .

  • In contrast, in immune cells, ARID5B has been characterized as a pro-inflammatory factor through stabilization of certain mRNAs .

• Protein interaction network mapping:

  • Use co-immunoprecipitation with ARID5B antibodies followed by mass spectrometry to identify cell-type-specific interaction partners.

  • The PHF2-ARID5B complex functions as a coactivator of HNF4A specifically in liver tissue .

  • In different cellular contexts, ARID5B may interact with distinct partners, explaining functional divergence.

• Chromatin and RNA occupancy profiling:

  • Perform ChIP-seq and CLIP-seq (or iCLIP2) with validated ARID5B antibodies across cell types to map binding sites.

  • ARID5B exhibits dual DNA/RNA binding capabilities, with DNA preference for AT-rich regions and RNA preference for CAGGCAG consensus motif and AU-rich regions .

  • Compare binding profiles between cell types to identify context-specific targets.

• Genetic perturbation with phenotypic readouts:

  • Implement isogenic cell models using CRISPR-Cas9 to introduce identical genetic alterations across cell types.

  • Measure relevant functional outputs (e.g., cytokine production, adipogenesis markers) after perturbation.

  • siRNA knockdown experiments have identified ARID5B among 7 transcription factors affecting IL-6 production in RASFs .

• Single-cell analysis to resolve cellular heterogeneity:

  • Use single-cell RNA-seq data to identify cell subpopulations with distinct ARID5B expression patterns.

  • Recent single-cell RNA-sequencing analysis of rheumatoid arthritis synovial tissues revealed heterogeneity of RA synovial fibroblasts with distinct functions such as high IL-6 production .

• Signaling pathway analysis:

  • Test how different stimuli affect ARID5B function across cell types.

  • In synovial fibroblasts, TNF-α negatively regulates both long and short ARID5B isoforms .

  • In immune cells, ARID5B shuttles between nucleus and cytoplasm upon lipopolysaccharide stimulation .

These complementary approaches can resolve apparent contradictions by revealing how cellular context, signaling environment, and isoform expression patterns collectively determine ARID5B function in different systems.

What emerging technologies could enhance ARID5B antibody-based research in the near future?

Several emerging technologies promise to advance ARID5B antibody-based research with increased precision and functional insight:

• Proximity-based labeling with antibody specificity:

  • Techniques like TurboID or APEX2 fused to nanobodies against ARID5B can map proximal protein interactions in living cells.

  • This approach could reveal dynamic, context-specific interactomes of ARID5B across different cell types and conditions.

  • Particularly valuable for understanding how ARID5B functions differently in nuclear versus cytoplasmic compartments during cellular responses .

• High-throughput antibody validation platforms:

  • Automated, multi-parameter assessment of antibody specificity across diverse samples and conditions.

  • Integration with machine learning algorithms to predict optimal antibody applications.

  • Especially important given the multiple isoforms of ARID5B and their differential functions .

• Single-cell protein-DNA/RNA interaction mapping:

  • Integration of antibody-based detection with single-cell multiomics to correlate ARID5B binding with transcriptional outcomes.

  • Given ARID5B's dual binding capabilities for DNA and RNA , these techniques could reveal cell-specific regulatory networks.

  • Could help explain the heterogeneity observed in rheumatoid arthritis synovial fibroblasts with distinct IL-6 production profiles .

• Spatial proteomics with ARID5B-specific antibodies:

  • Techniques like Imaging Mass Cytometry or CODEX with ARID5B antibodies can map protein localization in tissue contexts.

  • This approach could reveal microenvironmental influences on ARID5B function in complex tissues like synovium or bone marrow.

• CRISPR-based genetic screens with antibody readouts:

  • Pooled CRISPR screens targeting genes that interact with ARID5B, using antibody-based detection of ARID5B as a phenotypic readout.

  • This could identify novel regulators of ARID5B expression or function.

  • Similar approaches have already been applied to identify cis-regulatory elements affecting ARID5B expression in ALL cells .

• Computationally designed antibodies for isoform specificity:

  • AI-driven antibody design targeting unique epitopes of ARID5B isoforms.

  • This could overcome current limitations in distinguishing the functional roles of long versus short isoforms .

These technological advances could significantly enhance our understanding of ARID5B's complex roles in diverse cellular contexts and disease states, potentially leading to new therapeutic strategies targeting its activity.

How might ARID5B antibodies contribute to therapeutic development for conditions like rheumatoid arthritis?

ARID5B antibodies offer significant potential for therapeutic development in conditions like rheumatoid arthritis through several research pathways:

• Target validation and drug screening:

  • ARID5B has been identified as a negative modulator of IL-6 production in rheumatoid arthritis synovial fibroblasts (RASFs) .

  • Antibodies can be used to validate ARID5B as a druggable target and screen for compounds that modulate its activity.

  • Since the long isoform of ARID5B suppresses TNF-α-stimulated IL-6 production , isoform-specific antibodies can help identify which protein domain to target therapeutically.

• Biomarker development:

  • ARID5B expression levels detected by antibodies could serve as biomarkers for treatment response or disease progression.

  • The RA risk allele rs10821944 in ARID5B intron 4 affects expression of the long isoform specifically in TNF-α-treated synovial fibroblasts , suggesting genetic stratification possibilities.

  • Antibody-based assays could identify patients likely to benefit from ARID5B-targeted therapies.

• Therapeutic antibody engineering:

  • Developing antibodies that mimic or enhance ARID5B's negative regulation of IL-6 production.

  • Antibodies could be designed to specifically upregulate the long isoform of ARID5B, which has suppressible effects on inflammatory cytokine production .

• Antibody-drug conjugates:

  • ARID5B-targeting antibodies could deliver anti-inflammatory compounds directly to cells expressing high levels of ARID5B.

  • This approach could enhance therapeutic efficacy while reducing systemic side effects.

• Combination therapy optimization:

  • Antibody-based detection of ARID5B expression changes in response to existing RA treatments could guide combination therapy approaches.

  • Since TNF-α negatively regulates ARID5B expression , combining TNF inhibitors with ARID5B-enhancing compounds might have synergistic effects.

• Precision medicine applications:

  • eQTL analysis using 58 synovial fibroblasts demonstrated genetic associations with ARID5B isoform expression .

  • Antibody-based detection of patient-specific ARID5B expression patterns could guide personalized treatment approaches.

  • This is particularly relevant given the heterogeneity of RA synovial fibroblasts with distinct inflammatory profiles .

This multifaceted approach using ARID5B antibodies as research tools could accelerate the development of novel therapies for rheumatoid arthritis and potentially other inflammatory conditions, addressing an important unmet medical need for patients who remain refractory to current treatments .