UBE4A Antibody

What is UBE4A and what cellular functions does it perform?

UBE4A (ubiquitination factor E4A) is a U-box-type ubiquitin-protein ligase that functions as an E3 ligase, working in conjunction with specific E1 and E2 ligases in the ubiquitin-proteasome system . This 123-124 kDa protein (1073 amino acids) is encoded by the UBE4A gene (Gene ID: 9354) and plays critical roles in protein degradation pathways .

UBE4A participates in several key cellular processes:

• Targeting proteins for proteasomal degradation

• Mediating apoA-I ubiquitination and degradation in hepatocytes

• Working with IP6K1 via its product 5PP-InsP5 to regulate protein degradation

• Potentially contributing to disease processes when dysregulated, as observed in Crohn's disease

The cellular localization and tissue distribution studies indicate UBE4A is expressed in multiple cell types, with notable upregulation observed in enteroendocrine cells of inflamed ileal mucosa in Crohn's disease patients .

What are the recommended applications and dilutions for UBE4A antibodies in common research techniques?

UBE4A antibodies have been validated for several experimental applications, with specific recommendations for optimal results:

It is strongly recommended to optimize these dilutions for each specific experimental system, as detection sensitivity can vary based on sample type, protein expression levels, and detection methods . When implementing these techniques, researchers should also consider:

• Including proper positive and negative controls

• Using freshly prepared samples when possible

• Implementing gentle wash steps for IP applications to preserve protein-protein interactions

• Confirming specificity through knockdown/knockout validation

What are the critical parameters for validated UBE4A antibody storage and handling?

Proper storage and handling of UBE4A antibodies is essential for maintaining reactivity and specificity:

Storage conditions:

• Store at -20°C in aliquots to minimize freeze-thaw cycles

• The antibody remains stable for one year after shipment when properly stored

• For 21548-1-AP specifically, aliquoting is unnecessary for -20°C storage

• Some preparations (20μl sizes) contain 0.1% BSA as a stabilizer

Buffer composition impact:

• Standard formulation includes PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

• Avoid repeated freeze-thaw cycles which can lead to protein denaturation and loss of activity

• For long-term storage beyond one year, consider storing small aliquots at -80°C

When handling UBE4A antibodies for experiments, researchers should:

• Allow the antibody to equilibrate to room temperature before opening

• Centrifuge briefly before use to collect all material at the bottom of the tube

• Use sterile technique when handling to prevent contamination

• Record lot numbers and maintain consistency within experimental series for reproducibility

How can UBE4A antibodies be validated for specificity in knockout/knockdown systems?

Validating UBE4A antibodies through knockout/knockdown approaches is essential for confirming specificity and preventing misinterpretation of experimental results:

Recommended validation protocol:

• RNAi-based validation:

  • Transfect cells with UBE4A-specific siRNA or shRNA (at least 2-3 different sequences targeting different regions)

  • Confirm knockdown efficiency through RT-PCR (>70% reduction at mRNA level)

  • Perform Western blot using the UBE4A antibody, comparing knockdown samples to control siRNA/shRNA samples

  • A specific antibody will show significant reduction in band intensity at the expected molecular weight (123 kDa)

• CRISPR/Cas9 knockout validation:

  • Generate UBE4A knockout cell lines using CRISPR/Cas9 technology

  • Confirm genomic disruption through sequencing

  • Perform Western blot comparison between wild-type and knockout cells

  • Complete absence of the target band confirms specificity

• Overexpression validation:

  • Transfect cells with UBE4A expression vector (epitope-tagged version is recommended)

  • Perform Western blot using UBE4A antibody

  • Enhanced signal at expected molecular weight supports specificity

Research studies have demonstrated that proper UBE4A antibody validation successfully detects changes in UBE4A expression, as observed in studies examining UBE4A-mediated apoA-I ubiquitination and degradation . When UBE4A was deleted using shRNA in primary murine hepatocytes, researchers confirmed reduced UBE4A protein levels corresponded with decreased apoA-I ubiquitination, verifying both antibody specificity and functional effects simultaneously.

What are the optimal protocols for detecting UBE4A-protein interactions using co-immunoprecipitation?

Co-immunoprecipitation (co-IP) is a powerful technique for studying UBE4A's interactions with binding partners and substrates. Based on published methodologies, the following protocol has been optimized for UBE4A interaction studies:

Optimized UBE4A co-IP protocol:

• Cell preparation and lysis:

  • Harvest cells (primary hepatocytes or appropriate cell lines)

  • Lyse cells in buffer containing protease inhibitors

  • Pre-clean lysates with protein A/G beads to reduce non-specific binding

• Immunoprecipitation stage:

  • For tagged proteins: Use anti-tag antibody (e.g., anti-flag for flag-apoA-I) with protein A/G beads

  • For endogenous proteins: Use UBE4A-specific antibody (e.g., 21548-1-AP)

  • Incubate overnight at 4°C with gentle rotation

  • Wash beads at least three times with lysis buffer

• Detection and analysis:

  • Elute bound proteins with SDS loading buffer containing β-mercaptoethanol

  • Analyze by SDS-PAGE and Western blotting

  • Probe for potential interaction partners

This approach has successfully identified several UBE4A interactions, including:

• UBE4A binding to apoA-I (confirmed through reciprocal co-IPs)

• UBE4A interaction with IP6K1 (validated in both overexpression systems and with endogenous proteins)

• Regulation of these interactions by 5PP-InsP5

When optimizing co-IP for UBE4A studies, researchers should consider adjusting salt concentration in wash buffers to modulate stringency based on interaction strength.

How can immunohistochemistry protocols be optimized for UBE4A detection in tissue samples?

Immunohistochemistry (IHC) for UBE4A requires careful optimization to maximize specific signal while minimizing background. Based on published research, particularly studies examining UBE4A expression in intestinal tissues, the following approach is recommended:

Optimized UBE4A IHC protocol:

• Tissue preparation:

  • Fix tissues in 10% neutral buffered formalin (optimal fixation time depends on tissue thickness)

  • Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Block endogenous peroxidase activity with 3% hydrogen peroxide

  • Apply protein block (serum-free) to reduce non-specific binding

• Antibody incubation and detection:

  • Use UBE4A primary antibody at optimized dilution (typically 1:100-1:500)

  • Incubate overnight at 4°C in humidified chamber

  • Apply appropriate secondary antibody system

  • Develop with DAB chromogen and counterstain with hematoxylin

  • Mount and analyze

This approach has successfully detected differential expression of UBE4A in tissues, including upregulation in enteroendocrine cells of inflamed ileal mucosa in Crohn's disease patients . Cell-type specific expression patterns can provide valuable insights into UBE4A's role in tissue homeostasis and disease.

For double immunofluorescence staining to identify specific cell types expressing UBE4A, researchers should consider incorporating cell-type specific markers (e.g., chromogranin A for enteroendocrine cells) in the protocol.

What experimental approaches are recommended for studying UBE4A-mediated protein ubiquitination?

Investigating UBE4A's role in protein ubiquitination requires specialized techniques to capture and quantify this post-translational modification. Based on successful research methodologies, the following approaches are recommended:

In-cell ubiquitination assays:

• Transfect cells with constructs expressing UBE4A (wild-type and/or mutant variants)

• Co-transfect with tagged-ubiquitin (e.g., HA-ubiquitin) and potential substrate protein

• Treat cells with proteasome inhibitor (e.g., MG132) for 4-6 hours prior to lysis

• Immunoprecipitate the substrate protein under denaturing conditions

• Analyze ubiquitination by Western blot using anti-tag antibody

This approach has been successfully employed to demonstrate UBE4A-mediated ubiquitination of apoA-I. When UBE4A was overexpressed in primary murine hepatocytes, researchers observed increased apoA-I ubiquitination, while UBE4A knockdown resulted in decreased ubiquitination levels .

In vitro ubiquitination assays:
For more direct evidence of UBE4A's E3 ligase activity, researchers can:

• Purify recombinant UBE4A, E1, E2 enzymes, and potential substrate

• Combine components in reaction buffer with ATP and ubiquitin

• Incubate at 30°C for 1-2 hours

• Analyze reaction products by SDS-PAGE and Western blot

• Confirm ubiquitination through mass spectrometry analysis

When implementing these approaches, it is critical to include appropriate controls:

• Catalytically inactive UBE4A mutant

• Reaction without ATP (negative control)

• Reaction without E1 or E2 enzymes

How does UBE4A interact with the ubiquitin-proteasome pathway, and what methods best elucidate these mechanisms?

UBE4A functions within the broader ubiquitin-proteasome system (UPS), and understanding these interactions requires specialized experimental approaches:

Recommended methodologies for studying UBE4A-UPS interactions:

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (MG132, bortezomib)

  • Compare protein levels of UBE4A substrates with and without inhibition

  • Measure half-life changes of substrates with pulse-chase experiments

  Research has shown that UBE4A-targeted proteins accumulate when proteasome function is inhibited, confirming the role of proteasomal degradation in UBE4A substrate fate .

• E1/E2 interaction mapping:

  • Perform yeast two-hybrid or GST-pulldown assays to identify specific E2 enzymes working with UBE4A

  • Validate interactions through co-IP in mammalian cells

  • Test different E2 enzymes in in vitro ubiquitination assays to identify functional pairs

• Domain function analysis:

  • Generate UBE4A constructs with mutations in key domains:

    • U-box domain (critical for E3 ligase activity)

    • UBL domain (potential proteasome interaction)

    • Other protein-protein interaction domains

  • Assess effects on substrate ubiquitination and degradation

• Ubiquitin linkage analysis:

  • Use ubiquitin mutants (K48R, K63R, etc.) to determine which lysine residues are used for chain formation

  • Employ ubiquitin linkage-specific antibodies in Western blot

  • Apply mass spectrometry to identify ubiquitination sites and chain topology

Research has demonstrated that UBE4A can promote K48-linked polyubiquitination, typically associated with proteasomal targeting, making it a key player in protein homeostasis regulation .

What is the relationship between UBE4A and IP6K1 in protein degradation pathways?

Research has revealed a critical functional relationship between UBE4A and inositol hexakisphosphate kinase 1 (IP6K1) in regulating protein degradation, particularly for apolipoprotein A-I (apoA-I). This interaction represents an important regulatory mechanism in protein homeostasis:

Key aspects of the UBE4A-IP6K1 relationship:

• Complex formation:

  • IP6K1 forms a complex with both UBE4A and apoA-I

  • This interaction was confirmed through immunoprecipitation followed by mass spectrometry

  • Co-immunoprecipitation experiments validated the binding between these proteins in both overexpression systems and with endogenous proteins

• 5PP-InsP5-dependent regulation:

  • IP6K1 produces 5PP-InsP5, an inositol pyrophosphate

  • 5PP-InsP5 enhances the binding between UBE4A and apoA-I

  • When IP6K1 is deleted or inhibited (using SC-919), reduced 5PP-InsP5 levels lead to decreased UBE4A-apoA-I interaction

• Functional consequences:

  • In IP6K1 knockout hepatocytes, less UBE4A binds to apoA-I

  • This reduced interaction results in decreased apoA-I ubiquitination and degradation

  • Consequently, apoA-I protein levels increase both intracellularly and in secreted form

The table below summarizes experimental evidence for this regulatory mechanism:

This regulatory pathway has significant implications for lipid metabolism and atherosclerosis protection, as elevated apoA-I levels contribute to increased HDL-C and enhanced reverse cholesterol transport .

What is the significance of anti-UBE4A autoantibodies in Crohn's disease, and how are they detected?

Anti-UBE4A autoantibodies have emerged as potentially important biomarkers in Crohn's disease (CD), with significant implications for diagnosis, disease activity monitoring, and outcome prediction:

Clinical significance of anti-UBE4A autoantibodies:

• Diagnostic potential:

  • Anti-UBE4A IgG prevalence is significantly higher in CD patients (46.2%) compared to ulcerative colitis patients (7.1%) and healthy controls (3.3%)

  • This substantial difference (p<0.0001 vs. healthy controls) suggests utility as a disease-specific marker

• Correlation with disease parameters:

  • Anti-UBE4A antibody levels strongly correlate with disease activity (r = 0.777, p<0.0001)

  • Antibody positivity is associated with stricturing or penetrating disease phenotypes (p=0.0028)

  • Anti-UBE4A-positive CD patients have higher likelihood of requiring surgical intervention (p=0.0013)

Methodology for anti-UBE4A autoantibody detection:

The research protocol for detecting these autoantibodies involves:

• Antigen preparation:

  • Recombinant UBE4A protein expressed from cDNA clone

  • Expression in appropriate bacterial or mammalian systems

  • Purification via affinity chromatography

• ELISA-based detection:

  • Coat plates with purified UBE4A protein

  • Incubate with patient serum (typically diluted 1:100)

  • Detect bound antibodies using anti-human IgG-HRP conjugate

  • Develop with appropriate substrate and measure absorbance

  • Establish cut-off values based on healthy control samples

• Confirmation through other methods:

  • Western blot analysis using recombinant UBE4A

  • Immunofluorescence with UBE4A-expressing cells

These findings suggest potential clinical applications for anti-UBE4A antibody testing in distinguishing CD from ulcerative colitis, predicting disease severity, and potentially guiding treatment decisions .

How can UBE4A's role in protein degradation be investigated in disease models?

Investigating UBE4A's role in disease pathogenesis requires specialized approaches that connect its function in protein degradation to disease-relevant pathways. The following methodologies are recommended for disease-focused UBE4A research:

Cell culture disease models:

• Gene expression manipulation:

  • Overexpress or knockdown UBE4A in disease-relevant cell types

  • Assess effects on known disease-associated proteins

  • Measure changes in protein half-life using cycloheximide chase assays

  • Quantify ubiquitination levels of potential disease-related substrates

• Disease-specific stimulation:

  • Expose cells to disease-relevant stimuli (e.g., inflammatory cytokines for Crohn's disease models)

  • Analyze UBE4A expression, localization, and activity changes

  • Identify altered protein degradation pathways using proteomic approaches

Animal disease models:

• Tissue-specific UBE4A manipulation:

  • Generate conditional UBE4A knockout or overexpression animals

  • Target disease-relevant tissues using appropriate Cre-driver lines

  • Assess disease phenotypes and progression

• Disease model interventions:

  • Subject UBE4A-modified animals to established disease protocols

  • For inflammatory bowel disease: dextran sodium sulfate (DSS) or TNBS-induced colitis

  • For atherosclerosis: high-fat diet in ApoE-deficient background

  • Compare disease severity and progression between UBE4A-modified and control animals

• Therapeutic targeting assessment:

  • Test whether modulating UBE4A or its interaction partners (e.g., IP6K1) affects disease outcomes

  • Administer IP6K1 inhibitors like SC-919 and assess impacts on UBE4A-regulated proteins and disease parameters

Research has demonstrated that inhibiting IP6K1, which regulates UBE4A-mediated apoA-I degradation, confers atheroprotection and improves metabolic parameters, suggesting therapeutic potential in targeting this pathway .

What experimental approaches can determine if UBE4A dysfunction is causative in disease pathogenesis?

Establishing a causal relationship between UBE4A dysfunction and disease requires rigorous experimental approaches that go beyond correlation. The following methodologies can help determine whether UBE4A alterations directly contribute to disease pathogenesis:

Genetic evidence approaches:

• Human genetic studies:

  • Screen for UBE4A mutations or polymorphisms in patient cohorts

  • Perform association studies to link genetic variants with disease risk

  • Conduct functional analysis of disease-associated variants

• CRISPR-based disease modeling:

  • Introduce specific UBE4A mutations identified in patients into cells or animal models

  • Generate isogenic cell lines differing only in UBE4A status

  • Compare phenotypic outcomes focusing on disease-relevant parameters

Functional rescue experiments:

• Reconstitution studies:

  • Knockdown or knockout endogenous UBE4A

  • Rescue with wild-type or mutant UBE4A variants

  • Assess which UBE4A functions are required to prevent or reverse disease phenotypes

• Structure-function analysis:

  • Generate UBE4A constructs with mutations in specific functional domains:

    • U-box domain (E3 ligase activity)

    • Substrate binding regions

    • Protein-protein interaction domains

  • Determine which domains are essential for disease-relevant functions

Substrate identification and validation:

• Proteomics approaches:

  • Perform quantitative proteomics comparing control and UBE4A-manipulated samples

  • Identify proteins with altered abundance or ubiquitination status

  • Focus on disease-relevant pathways

• Direct substrate validation:

  • Confirm UBE4A-substrate interactions using co-immunoprecipitation

  • Verify direct ubiquitination using in vitro and in vivo ubiquitination assays

  • Demonstrate biological consequences of substrate regulation

Research on UBE4A in Crohn's disease has shown that UBE4A is upregulated in enteroendocrine cells of inflamed ileal mucosa, suggesting its involvement in the inflammatory process . Similarly, studies in atherosclerosis models have demonstrated that inhibiting UBE4A-mediated apoA-I degradation (through IP6K1 inhibition) confers atheroprotection, establishing a mechanistic link between this pathway and disease progression .

How can researchers design experiments to identify novel UBE4A substrates and interaction partners?

Identifying novel UBE4A substrates and interaction partners requires strategic experimental approaches that leverage both targeted and unbiased methods:

Unbiased proteomic approaches:

• BioID or proximity labeling:

  • Generate UBE4A fusion with BioID2 or TurboID biotin ligase

  • Express in relevant cell types and activate biotinylation

  • Purify biotinylated proteins using streptavidin

  • Identify candidates via mass spectrometry

  • This approach captures both stable and transient interactors

• Quantitative ubiquitinome analysis:

  • Compare ubiquitinated protein profiles between control and UBE4A-manipulated cells

  • Employ tandem ubiquitin binding entities (TUBEs) to enrich ubiquitinated proteins

  • Use SILAC or TMT labeling for quantitative comparison

  • Identify proteins with decreased ubiquitination upon UBE4A depletion

• Co-immunoprecipitation coupled with mass spectrometry:

  • Immunoprecipitate UBE4A from relevant cell types or tissues

  • Identify co-precipitated proteins by mass spectrometry

  • Compare results under different cellular conditions (e.g., stress, differentiation)

Candidate-based approaches:

• Domain-based prediction:

  • Analyze protein regions/motifs recognized by UBE4A

  • Screen for proteins containing similar motifs

  • Validate interaction through co-immunoprecipitation

  • Test ubiquitination in cell-based and in vitro assays

• Yeast two-hybrid screening:

  • Use UBE4A or specific domains as bait

  • Screen against cDNA libraries from tissues of interest

  • Validate positive interactions in mammalian cells

Validation pipeline for candidate substrates:

This comprehensive approach has successfully identified apoA-I as a UBE4A substrate, demonstrating that UBE4A mediates its ubiquitination and subsequent degradation .

What controls and validation steps are essential when publishing UBE4A research findings?

Publishing rigorous UBE4A research requires comprehensive controls and validation steps to ensure reproducibility and reliability of findings:

Essential experimental controls:

• Antibody validation controls:

  • Include UBE4A knockout/knockdown samples to confirm specificity

  • Demonstrate antibody reactivity with overexpressed UBE4A

  • Present full, uncut Western blot images with molecular weight markers

  • Validate across multiple techniques (WB, IP, IF/IHC) when possible

• Functional assay controls:

  • For ubiquitination assays: include negative controls (without E1, E2, or ATP)

  • For degradation studies: include proteasome inhibitor conditions

  • For siRNA/shRNA experiments: include non-targeting control and rescue experiments

  • For CRISPR experiments: use multiple guide RNAs and validate editing

• Expression manipulation controls:

  • For overexpression: compare multiple expression levels and empty vector controls

  • For knockdown: test multiple siRNA/shRNA sequences to rule out off-target effects

  • For both: confirm specificity by measuring effects on closely related proteins

Critical validation steps:

• Mechanistic validation:

  • Demonstrate direct UBE4A-substrate interaction through multiple methods

  • Show direct ubiquitination in both cellular and in vitro systems

  • Identify ubiquitination sites through mutagenesis or mass spectrometry

  • Connect ubiquitination to functional outcomes (degradation, localization, activity)

• Physiological relevance:

  • Validate findings in primary cells and/or relevant tissues

  • Demonstrate regulation under physiological conditions

  • Connect to known disease mechanisms when applicable

  • Show conservation across species when relevant

• Reproducibility measures:

  • Report exact experimental conditions, reagent sources, and cell line authentication

  • Include statistical analysis with appropriate tests and multiple biological replicates

  • Validate key findings with alternative methodologies

  • Consider independent validation in different cell types or model systems

Following these validation guidelines has been critical in establishing UBE4A's role in diverse processes, including its connection to apoA-I degradation and atheroprotection , and its potential involvement in Crohn's disease pathogenesis .

How can researchers integrate UBE4A findings with other ubiquitination pathways in experimental design?

UBE4A functions within a complex network of ubiquitination pathways, and integrative experimental approaches are needed to understand its unique and overlapping roles:

Pathway integration approaches:

• E3 ligase substrate comparison:

  • Identify substrates targeted by multiple E3 ligases including UBE4A

  • Determine whether different E3s target the same or different lysine residues

  • Assess cooperation or competition between UBE4A and other E3 ligases

  • Example: Compare UBE4A-mediated ubiquitination with other E3s known to target the same substrate

• Deubiquitinase (DUB) interaction studies:

  • Screen for DUBs that counteract UBE4A-mediated ubiquitination

  • Examine UBE4A substrate stability when relevant DUBs are inhibited

  • Investigate potential direct interaction between UBE4A and specific DUBs

  • Example: Test whether USP family members can deubiquitinate UBE4A targets

• Pathway crosstalk analysis:

  • Investigate how UBE4A function is affected by other post-translational modifications

  • Examine how UBE4A activity affects or is affected by related pathways:

    • Other degradation pathways (autophagy, lysosomal)

    • Stress response pathways (unfolded protein response, heat shock)

    • Cell cycle regulation

  • Example: Study how ER stress affects UBE4A-mediated apoA-I degradation

Integrative experimental strategies:

• Multi-omics approach:

  • Combine proteomics, transcriptomics, and ubiquitinomics data

  • Correlate changes in UBE4A expression/activity with global pathway alterations

  • Identify key nodes where UBE4A intersects with other cellular pathways

  • Example: Compare proteome and ubiquitinome changes in UBE4A-manipulated cells

• Network analysis:

  • Map UBE4A interactions and substrates within broader protein interaction networks

  • Identify protein complexes or functional modules containing UBE4A

  • Determine whether UBE4A targets specific cellular compartments or processes

  • Example: Place UBE4A-IP6K1-apoA-I interactions in the context of lipid metabolism networks

• Systems biology modeling:

  • Develop computational models incorporating UBE4A activity

  • Simulate effects of UBE4A perturbation on cellular homeostasis

  • Predict compensatory mechanisms and pathway adaptations

  • Example: Model how changes in UBE4A activity affect protein turnover dynamics

Research has demonstrated that UBE4A functions in concert with IP6K1 via its product 5PP-InsP5 to regulate apoA-I degradation, illustrating how UBE4A activity is integrated with other signaling pathways . This integrative approach has revealed that inhibiting IP6K1 confers atheroprotection by elevating apoA-I levels, highlighting the therapeutic potential of targeting these interconnected pathways.