UBASH3A Antibody, Biotin conjugated

What is UBASH3A and what is its primary function in cellular pathways?

UBASH3A (Ubiquitin Associated and SH3 Domain Containing A), also known as T-cell ubiquitin ligand 1 (TULA-1) or Suppressor of T-cell receptor signaling 2 (STS-2), is a protein primarily expressed in T cells that functions as a negative regulator of T-cell signaling. The protein contains multiple functional domains including an SH3 domain and a ubiquitin-binding domain that enable its regulatory activity. UBASH3A attenuates NF-κB signal transduction upon T-cell receptor (TCR) stimulation by specifically suppressing the activation of the IκB kinase complex. This regulation occurs through novel interactions with nondegradative polyubiquitin chains, TAK1, and NEMO, suggesting that UBASH3A modulates the NF-κB signaling pathway through an ubiquitin-dependent mechanism . Additionally, UBASH3A can facilitate growth factor withdrawal-induced apoptosis in T cells via its interaction with the apoptosis-inducing factor (AIF) . The protein is also known to associate with c-Cbl and ubiquitylated proteins, further implicating its role in the regulation of signaling mediated by protein-tyrosine kinases .

What is the tissue distribution and expression pattern of UBASH3A?

UBASH3A expression is limited to specific tissues, with highest expression observed in lymphoid organs and cells. Based on comprehensive tissue expression profiling, UBASH3A shows predominant expression in:

• Spleen

• Peripheral blood leukocytes

• Bone marrow

This restricted expression pattern aligns with UBASH3A's specialized function in immune cell regulation, particularly T-cell signaling. The protein exhibits dual subcellular localization, being present in both the cytoplasm and nucleus , which suggests multiple functional roles depending on cellular compartmentalization. This expression profile makes UBASH3A particularly relevant for immunological research and studies focused on T-cell biology, autoimmune disorders, and lymphoid-derived pathologies.

How does a biotin-conjugated UBASH3A antibody differ from unconjugated versions?

Biotin-conjugated UBASH3A antibodies differ from unconjugated versions primarily in their detection capabilities and experimental applications. The biotin conjugation involves chemical linkage of biotin molecules to the antibody structure, which provides several functional advantages:

• Detection sensitivity: Biotin-conjugated antibodies leverage the strong avidin-biotin interaction (one of the strongest non-covalent biological interactions), enabling signal amplification for enhanced detection sensitivity compared to unconjugated antibodies.

• Visualization flexibility: The biotin tag allows for multiple detection strategies using streptavidin or avidin conjugated to various reporter molecules (HRP, fluorophores, gold particles), providing versatility in experimental design.

• Multi-step detection protocols: Biotin-conjugated antibodies are particularly valuable in multi-layered staining protocols where direct detection might be challenging.

• Storage stability: The biotin-conjugated UBASH3A antibody (as seen in product TA809272AM) is stored in PBS containing 1% BSA, 50% glycerol, and 0.02% sodium azide at -20°C, which differs from some unconjugated versions .

For research applications specifically targeting UBASH3A, biotin conjugation provides technical advantages in detection workflows without altering the specificity of the antibody (still targeting human UBASH3A as seen in the mouse monoclonal antibody clone OTI5B4) .

What are the recommended applications for biotin-conjugated UBASH3A antibodies?

Biotin-conjugated UBASH3A antibodies are versatile reagents suitable for multiple experimental applications, with specific recommendations based on validated protocols:

The biotin-conjugated format is particularly advantageous for signal amplification in detection systems using streptavidin-conjugated reporters. For optimal results, researchers should perform titration experiments to determine the ideal concentration for their specific experimental conditions, as sensitivity and background may vary depending on sample type and detection method .

What is the recommended protocol for using biotin-conjugated UBASH3A antibody in Western blotting?

For optimal Western blot results using biotin-conjugated UBASH3A antibody, follow this validated protocol:

• Sample preparation:

  • Prepare cell/tissue lysates in RIPA buffer containing protease inhibitors

  • For T-cell studies, stimulated and unstimulated controls are recommended

  • Use 20-40 μg of total protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on 10% SDS-PAGE gel

  • Transfer to PVDF membrane (0.45 μm pore size)

  • Confirm transfer efficiency with reversible protein stain

• Blocking and antibody incubation:

  • Block membrane in 5% non-fat milk in TBST for 1 hour at room temperature

  • Incubate with biotin-conjugated UBASH3A antibody (clone OTI5B4) at 1:2000 dilution in blocking buffer overnight at 4°C

  • Wash 3x with TBST, 5 minutes each

• Detection:

  • Incubate with streptavidin-HRP (1:5000) for 1 hour at room temperature

  • Wash 3x with TBST, 5 minutes each

  • Develop using enhanced chemiluminescence substrate

  • Expected molecular weight: 73.9 kDa

• Controls and validation:

  • Include positive control (lymphoid tissue lysate)

  • Include negative control (tissue with low UBASH3A expression)

  • Consider running UBASH3A knockout/overexpression samples as specificity controls

This protocol has been optimized based on experimental validation with human samples, but may require adjustment for mouse or rat samples depending on cross-reactivity .

How can I optimize immunohistochemistry protocols using UBASH3A antibodies?

Optimizing immunohistochemistry (IHC) protocols for UBASH3A detection requires careful attention to several key parameters:

• Tissue preparation and antigen retrieval:

  • Use freshly fixed tissues (10% neutral buffered formalin, 24-48 hours)

  • For FFPE samples, optimal heat-induced epitope retrieval uses citrate buffer (pH 6.0) for 20 minutes

  • For lymphoid tissues, protease-based retrieval may improve signal-to-noise ratio

• Antibody concentration optimization:

  • Start with manufacturer's recommended dilution range (1:40-1:250)

  • Perform serial dilutions to identify optimal concentration

  • For biotin-conjugated antibodies, implement additional streptavidin blocking to reduce endogenous biotin interference

• Detection system selection:

  • For biotin-conjugated UBASH3A antibodies, use streptavidin-HRP systems

  • ABC (Avidin-Biotin Complex) method provides signal amplification for low-abundance targets

  • Consider tyramide signal amplification for detecting minimal UBASH3A expression

• Validated positive controls:

  • Human tonsil (lymphoid follicles show strong positive staining)

  • Human bone marrow

  • Spleen sections (particularly T-cell zones)

• Counter-staining and visualization:

  • Hematoxylin counterstain (Mayer's formulation) for 30-60 seconds

  • For co-localization studies, combine with CD3 or other T-cell markers

Researchers should note that UBASH3A shows both cytoplasmic and nuclear localization , so evaluation of staining patterns should consider both compartments. Validation using multiple antibody clones or orthogonal detection methods is recommended for confirming specificity, particularly in complex tissue environments.

How does UBASH3A influence T-cell activation and signaling?

UBASH3A functions as a critical negative regulator of T-cell activation through multiple molecular mechanisms:

• NF-κB pathway suppression: UBASH3A specifically attenuates NF-κB signal transduction following T-cell receptor (TCR) stimulation by suppressing the activation of the IκB kinase complex. This regulatory activity involves novel interactions with nondegradative polyubiquitin chains, TAK1, and NEMO, suggesting an ubiquitin-dependent mechanism .

• Dose-dependent IL-2 regulation: Experimental evidence from gene knockout and overexpression studies demonstrates that UBASH3A levels inversely correlate with IL-2 production. UBASH3A knockout T-cell lines show significantly increased IL-2 production (two- to eight-fold higher) upon stimulation with either anti-CD3 alone or anti-CD3 plus anti-CD28. Conversely, overexpression of UBASH3A results in attenuated IL-2 production, with a clear dose-dependent relationship observed .

• pH-sensitive phosphatase activity: UBASH3A forms an STS1-Cbl-b complex with pH-sensitive phosphatase activity that suppresses T-cell function specifically in acidic environments . This mechanism may be particularly relevant in inflammatory microenvironments where pH changes occur.

• Apoptosis regulation: UBASH3A can facilitate growth factor withdrawal-induced apoptosis in T cells via interaction with apoptosis-inducing factor (AIF) . This suggests a role in T-cell homeostasis and population control.

These molecular activities position UBASH3A as a key checkpoint in T-cell activation, potentially contributing to the prevention of autoimmunity and hyperinflammatory responses through multiple signaling nodes.

What is the relationship between UBASH3A and autoimmune diseases?

UBASH3A has emerged as an important genetic risk factor for multiple autoimmune conditions through its regulation of T-cell signaling and activation. Research has revealed several key aspects of this relationship:

• Type 1 Diabetes (T1D) association: Genetic variants in UBASH3A are strongly associated with T1D risk. Specifically, risk alleles at rs11203203 and rs80054410 increase UBASH3A expression and decrease IL-2 expression in activated human primary CD4+ T cells . This establishes a direct mechanistic link between genetic variation, UBASH3A expression levels, and T-cell function relevant to autoimmunity.

• Molecular mechanism: The increased risk of autoimmunity appears to be mediated through UBASH3A's inhibition of T-cell activation and IL-2 production. When UBASH3A expression is elevated due to risk alleles, T-cell activation is dampened, potentially disrupting normal immune regulation and tolerance mechanisms .

• Multiple autoimmune diseases: Beyond T1D, UBASH3A variants have been implicated in several other autoimmune disorders, suggesting a common pathogenic mechanism involving T-cell dysregulation.

• Functional studies evidence: Experimental models demonstrate that modulating UBASH3A expression directly impacts T-cell function, with knockout models showing enhanced T-cell activation and IL-2 production, while overexpression models show suppressed T-cell responses .

These findings position UBASH3A as both a biomarker for autoimmune disease risk and a potential therapeutic target. Researchers working with UBASH3A antibodies can leverage these connections to investigate disease mechanisms and potential interventions targeting this pathway in autoimmune conditions.

How can UBASH3A antibodies be used to study ubiquitination processes?

UBASH3A antibodies provide valuable tools for investigating ubiquitination processes due to the protein's intrinsic ubiquitin-binding properties and regulatory role in ubiquitin-dependent signaling pathways. Researchers can employ several methodological approaches:

• Co-immunoprecipitation studies: UBASH3A antibodies can effectively isolate UBASH3A protein complexes containing ubiquitinated proteins. In experimental settings, 500 μg of whole-cell lysate from cells expressing UBASH3A can be incubated with anti-UBASH3A antibodies (or anti-tag antibodies for tagged versions), followed by protein G Dynabeads capture. This approach has successfully demonstrated UBASH3A's interaction with specific ubiquitin chains .

• Ubiquitin chain binding assays: After immunoprecipitating UBASH3A, researchers can test its binding affinity to specific ubiquitin oligomers (K48-linked, K63-linked, etc.) to characterize the specificity of ubiquitin recognition. This method has revealed preferential binding patterns important for UBASH3A's regulatory function .

• Analysis of UBASH3A monoubiquitination: Western blotting with UBASH3A antibodies can reveal the monoubiquitinated form of UBASH3A itself, appearing as a higher molecular weight band. This has been observed in experimental systems like the V5-tagged UBASH3A clones (1F6, 2F5, and 5E4), providing insight into UBASH3A's own regulation .

• Ubiquitination dynamics during T-cell activation: Using biotin-conjugated UBASH3A antibodies in combination with ubiquitin antibodies allows tracking of changes in ubiquitination patterns during T-cell receptor stimulation, revealing the temporal dynamics of these regulatory processes.

These approaches can be particularly valuable for studying how UBASH3A's interactions with ubiquitinated proteins contribute to the regulation of T-cell signaling pathways and potentially to autoimmune disease mechanisms.

How do genetic variants of UBASH3A affect antibody selection and experimental design?

Genetic variants of UBASH3A present important considerations for antibody selection and experimental design that can significantly impact research outcomes:

• Epitope accessibility in variant proteins:

  • Disease-associated variants like rs11203203 and rs80054410 may alter protein conformation or post-translational modifications

  • Antibodies targeting regions affected by these variations might show differential binding affinity

  • For comprehensive studies, select antibodies targeting conserved epitopes (like the OTI5B4 clone targeting amino acids 288-549)

• Expression level considerations:

  • Risk alleles are associated with increased UBASH3A expression

  • Experimental designs should account for variable expression levels

  • Standard curves with recombinant protein should be included for accurate quantification

• Detection of variant-specific effects:

  • When studying specific variants, consider paired antibodies:

    • One targeting the variant region

    • Another targeting a conserved region as control

• Cell type-specific expression patterns:

  • Genetic variants may alter tissue-specific expression patterns

  • Experimental designs should include appropriate positive controls

  • Consider flow cytometry to assess expression in specific immune cell subsets

• Functional readouts:

  • Include IL-2 production assays as functional readouts

  • Measure NF-κB pathway activation as surrogate for UBASH3A function

  • Compare results between cells with different UBASH3A genotypes

These considerations are particularly important for researchers investigating the relationship between UBASH3A genetic variants and autoimmune disease susceptibility, as the antibody selection can significantly affect the ability to detect relevant differences in protein expression and function.

What are the key considerations for multiplex assays incorporating UBASH3A antibodies?

Implementing successful multiplex assays with UBASH3A antibodies requires careful planning and optimization of several technical parameters:

• Antibody compatibility and cross-reactivity:

  • Validate that biotin-conjugated UBASH3A antibodies don't cross-react with other targets in your panel

  • Test for potential steric hindrance when multiple antibodies target proximal epitopes

  • Consider using UBASH3A antibodies from different host species than other targets in your panel

• Signal separation strategies:

  • For fluorescence-based multiplex assays, pair biotin-conjugated UBASH3A antibodies with streptavidin conjugates spectrally distant from other fluorophores

  • In chromogenic multiplexing, use distinct chromogens with good spectral separation

  • Sequential detection may be necessary if targets are co-localized

• Target abundance normalization:

  • UBASH3A is expressed at varying levels in different cell populations

  • Adjust antibody concentrations to balance signals for co-detection with high-abundance proteins

  • Include calibration controls for each target in the multiplex panel

• Multiplex protocol optimization:

  • For co-detection with phospho-specific antibodies, optimize fixation conditions (critical for signaling studies)

  • Test blocking reagents for minimal background across all detection channels

  • Validate that signal amplification steps don't cause channel bleed-through

• Technical validation approach:

  • Perform single-staining controls alongside multiplex assays

  • Include compensation controls for fluorescence-based systems

  • Verify co-localization patterns using confocal microscopy

These considerations are particularly important when designing experiments to study UBASH3A interactions with other signaling molecules in the T-cell activation pathway or when investigating its relationship with ubiquitinated proteins in complex signaling networks.

How can phosphorylation status of UBASH3A be analyzed in combination with antibody-based detection?

Analyzing UBASH3A phosphorylation status requires sophisticated methodological approaches that combine antibody-based detection with phosphorylation-specific techniques:

This multi-faceted approach enables researchers to correlate UBASH3A phosphorylation status with its functional roles in T-cell signaling regulation and potentially identify new therapeutic intervention points for autoimmune disorders.

What are common troubleshooting strategies for weak or non-specific signals with UBASH3A antibodies?

When encountering weak or non-specific signals with UBASH3A antibodies, researchers should implement systematic troubleshooting strategies:

• For weak signal issues:

  • Increase antibody concentration incrementally (starting from 1:2000 for Western blot )

  • Extend primary antibody incubation time (overnight at 4°C often improves signal)

  • Implement signal amplification systems (use high-sensitivity detection reagents)

  • Enhance sample preparation to preserve UBASH3A (add phosphatase and protease inhibitors)

  • Increase protein loading (UBASH3A is tissue-specific with variable expression levels )

• For non-specific binding:

  • Optimize blocking conditions (test alternative blockers like 5% BSA instead of milk)

  • Increase washing stringency (add 0.1% SDS or 0.5M NaCl to wash buffers)

  • Pre-absorb the antibody with non-specific proteins

  • Lower antibody concentration while extending incubation time

  • Use more specific detection systems (try monoclonal instead of polyclonal antibodies)

• Special considerations for biotin-conjugated antibodies:

  • Block endogenous biotin with avidin/biotin blocking kits

  • Minimize exposure to free biotin in buffers

  • Use streptavidin conjugates with lower background (specially purified versions)

  • Implement additional blocking steps for endogenous biotin-binding proteins

• Tissue-specific considerations:

  • For non-lymphoid tissues, use tissue-specific optimized protocols

  • Longer antigen retrieval times may be necessary for FFPE samples

  • For tissues with low UBASH3A expression, consider more sensitive detection methods

• Validation approaches:

  • Run parallel experiments with multiple UBASH3A antibodies targeting different epitopes

  • Include known positive controls (human lymphoid tissues )

  • Consider using UBASH3A knockout or overexpressing cell lines as specificity controls

These strategies should be implemented systematically, changing one variable at a time to identify the optimal conditions for your specific experimental system.

How can researchers validate the specificity of UBASH3A antibodies in their experimental systems?

Validating antibody specificity is crucial for ensuring reliable research outcomes. For UBASH3A antibodies, researchers should implement a multi-faceted validation approach:

• Genetic validation approaches:

  • Use CRISPR/Cas9-generated UBASH3A knockout cell lines as negative controls

  • Compare staining patterns in UBASH3A-overexpressing vs. wild-type cells

  • Utilize siRNA knockdown to demonstrate signal reduction correlating with protein depletion

  • Available UBASH3A knockout clones (like 2.1D6 and 2.1F7) can serve as valuable controls

• Immunoblot validation markers:

  • Verify detection of the correct molecular weight band (73.9-74 kDa)

  • Confirm detection of both unmodified and monoubiquitinated forms

  • Run multiple antibodies targeting different epitopes to confirm consistent detection

  • Use reducing and non-reducing conditions to assess potential conformational epitopes

• Tissue expression pattern validation:

  • Verify highest expression in spleen, peripheral blood leukocytes, and bone marrow

  • Confirm low/absent expression in non-lymphoid tissues

  • Compare with published RNA expression databases (e.g., GTEx, Human Protein Atlas)

  • Validate subcellular localization (cytoplasmic and nuclear)

• Peptide competition assays:

  • Pre-incubate antibody with immunizing peptide before application

  • Confirm signal reduction with increasing peptide concentration

  • Include irrelevant peptide controls to demonstrate specificity

• Functional correlation validation:

  • Correlate antibody staining intensity with functional readouts (IL-2 production)

  • Verify inverse relationship between UBASH3A detection and T-cell activation markers

  • Confirm expected changes in detection following T-cell receptor stimulation

Implementation of these validation strategies ensures that experimental findings accurately reflect UBASH3A biology rather than antibody artifacts, particularly important for studies investigating UBASH3A's role in T-cell signaling and autoimmune disease mechanisms.

What quality control measures ensure optimal performance of biotin-conjugated UBASH3A antibodies?

Maintaining optimal performance of biotin-conjugated UBASH3A antibodies requires rigorous quality control measures throughout storage, handling, and experimental use:

• Storage and stability monitoring:

  • Store antibodies at -20°C as recommended, avoiding repeated freeze-thaw cycles

  • Aliquot antibodies upon receipt to minimize freeze-thaw degradation

  • Monitor expiration dates (typical stability is one year when properly stored)

  • Validate activity of older antibody lots against fresh lots before critical experiments

• Pre-experimental validation:

  • Perform titration experiments with each new lot to confirm optimal working dilution

  • Test biotin conjugation efficiency using streptavidin binding assays

  • Verify specificity using positive control samples (lymphoid tissues)

  • Assess background levels in negative control samples

• Conjugation stability assessment:

  • Monitor for potential signs of biotin hydrolysis (decreasing signal over time)

  • Check for antibody aggregation prior to use (centrifuge briefly before pipetting)

  • For long-term storage, add stabilizing proteins (BSA) if not already present

  • Record lot-to-lot variability in conjugation efficiency

• Application-specific quality controls:

  • For Western blotting: include biotin blocking controls to assess endogenous biotin

  • For IHC/ICC: implement avidin/biotin blocking steps to minimize background

  • For flow cytometry: use fluorescence-minus-one (FMO) controls for accurate gating

  • For all applications: include isotype controls with matching biotin conjugation

• Documentation and standardization:

  • Maintain detailed records of antibody performance across experiments

  • Standardize protocols to minimize technical variability

  • Create internal reference standards for consistent quantification

  • Document biotin-conjugated antibody performance metrics (signal-to-noise ratio)

These quality control measures are essential for generating reproducible and reliable data, particularly important for studies involving UBASH3A's complex roles in T-cell signaling and autoimmune disease mechanisms where subtle changes in protein expression or modification status can have significant biological implications.

How might UBASH3A antibodies contribute to therapeutic development for autoimmune diseases?

UBASH3A antibodies represent valuable tools for developing novel therapeutic strategies targeting autoimmune diseases through several research pathways:

• Target validation studies:

  • Biotin-conjugated UBASH3A antibodies enable precise quantification of UBASH3A expression in patient samples

  • Correlation of UBASH3A levels with disease severity provides validation for therapeutic targeting

  • Monitoring changes in UBASH3A expression following current immunomodulatory treatments helps identify mechanistic connections

• Screening platforms for drug discovery:

  • High-throughput screening assays using UBASH3A antibodies can identify compounds that modulate its expression or function

  • Proximity-based assays (like BRET or FRET) incorporating labeled UBASH3A antibodies can detect interaction disruptors

  • Cell-based reporter systems monitored with UBASH3A antibodies enable functional screening

• Biomarker development:

  • UBASH3A antibodies can be used to develop diagnostic or prognostic assays, particularly given the association of specific UBASH3A variants with disease risk

  • Monitoring UBASH3A expression or phosphorylation status might predict treatment response

  • Changes in UBASH3A cellular distribution (detected via fractionation and immunoblotting) could serve as disease activity indicators

• Therapeutic antibody development:

  • Research-grade antibodies provide valuable epitope information for developing therapeutic antibodies

  • Function-modulating antibodies targeting UBASH3A could potentially enhance T-cell responses in immunodeficiency or suppress them in autoimmunity

  • Intrabody approaches targeting UBASH3A could modulate T-cell function from within

• Combination therapy rationale:

  • UBASH3A antibodies help map signaling interactions, identifying potential synergistic targets

  • Studies of UBASH3A in the context of the STS1-Cbl-b complex suggest targeting multiple components might enhance efficacy

These research directions represent promising avenues for translating basic UBASH3A biology into clinical applications, potentially leading to more targeted therapies for autoimmune conditions like Type 1 Diabetes where UBASH3A genetic variants confer significant disease risk .

What emerging technologies might enhance UBASH3A detection and functional analysis?

Emerging technologies are poised to revolutionize UBASH3A research by enabling more sensitive detection, higher resolution analysis, and more comprehensive functional characterization:

• Single-cell proteomics approaches:

  • Mass cytometry (CyTOF) with UBASH3A antibodies allows simultaneous measurement of UBASH3A alongside dozens of other proteins at single-cell resolution

  • Imaging mass cytometry combines spatial information with high-parameter protein detection

  • Digital spatial profiling enables quantitative measurement of UBASH3A in tissue microenvironments

• Super-resolution microscopy applications:

  • STORM/PALM microscopy with biotin-conjugated UBASH3A antibodies enables nanoscale visualization of protein distribution

  • Lattice light-sheet microscopy allows dynamic tracking of UBASH3A translocation during T-cell activation

  • Expansion microscopy physically enlarges samples for enhanced resolution of protein complexes

• Proximity labeling technologies:

  • TurboID or APEX2 fusions to UBASH3A enable proximity biotinylation of interaction partners

  • Subsequently, biotin-conjugated UBASH3A antibodies can be used to validate these interactions

  • This approach maps the dynamic UBASH3A interactome during T-cell activation states

• CRISPR-based functional genomics:

  • CRISPR activation/inhibition screens targeting UBASH3A regulatory elements

  • Base editing to introduce specific disease-associated variants

  • CRISPR knock-in of tagged UBASH3A for live-cell imaging without antibody limitations

• Computational approaches:

  • Machine learning algorithms to analyze complex UBASH3A staining patterns

  • Integrative multi-omics approaches combining UBASH3A antibody-based proteomics with transcriptomics

  • Molecular modeling of UBASH3A interactions guided by antibody epitope mapping

These technologies will provide unprecedented insights into UBASH3A biology, particularly its dynamic regulation during T-cell activation and its dysregulation in autoimmune conditions. Researchers can leverage these advanced approaches to move beyond static measurements toward understanding the dynamic, contextual functions of UBASH3A in health and disease.

How can researchers best integrate UBASH3A antibody-based studies with genomic and transcriptomic data?

Integrating UBASH3A antibody-based studies with genomic and transcriptomic data creates powerful multi-omics approaches that provide comprehensive insights into UBASH3A biology:

• Genotype-phenotype correlation studies:

  • Quantify UBASH3A protein levels using calibrated antibody-based assays in samples with known genotypes at risk loci (rs11203203, rs80054410)

  • Correlate protein expression with risk alleles to validate functional consequences

  • Create integrated datasets combining:

    • UBASH3A protein levels (antibody-based detection)

    • Genotype at risk loci (genomic data)

    • Clinical phenotypes (patient metadata)

• Expression quantitative trait loci (eQTL) validation:

  • Use UBASH3A antibodies to confirm protein-level effects of eQTL variants

  • Compare mRNA expression (RNA-seq) with protein abundance (quantitative immunoblotting)

  • Identify post-transcriptional regulatory mechanisms by examining discordance between mRNA and protein levels

• Alternative splicing assessment:

  • Design epitope-specific antibodies targeting different UBASH3A isoforms

  • Correlate isoform-specific protein detection with RNA-seq splice variant quantification

  • Map functional consequences of different isoforms using antibody-based functional assays

• Multi-omics experimental design:

  • Collect matched samples for:

    • Genomic DNA (for genotyping)

    • RNA (for transcriptome analysis)

    • Protein (for antibody-based UBASH3A quantification)

    • Functional readouts (IL-2 production, T-cell activation)

  • Implement integrative analytical frameworks to identify concordant signals

• Technical integration approaches:

  • Spatial transcriptomics combined with UBASH3A immunofluorescence on the same tissue sections

  • Single-cell proteogenomics pairing UBASH3A antibody detection with single-cell RNA-seq

  • Chromatin immunoprecipitation (ChIP-seq) using UBASH3A antibodies to identify potential regulatory interactions

These integrative approaches enable researchers to bridge the gap between genetic association and molecular mechanism, particularly important for understanding how UBASH3A variants contribute to autoimmune disease risk through altered protein expression and function in specific cellular contexts.