UBE2D3 Antibody

What is UBE2D3 and why is it significant in research?

UBE2D3, also known as UBCH5C, is a member of the UBE2D (UBCH5) family of E2 ubiquitin-conjugating enzymes. It serves as a critical component in the ubiquitin-proteasome system by facilitating the transfer of ubiquitin to substrate proteins in conjunction with E3 ligases. UBE2D3's significance in research stems from its involvement in multiple important cellular processes including p53 regulation, DNA repair mechanisms, and protein quality control .

UBE2D3 has been linked to various pathological conditions, including multiple cancer types (breast, ovarian, cervical, head and neck, esophageal cancer, melanoma, leukemia, and multiple myeloma) as demonstrated by analyses of Oncomine, EMBL-EBI Expression Atlas, Cosmic, and ICGC databases . Recent studies have also established UBE2D3's role in cardiovascular pathologies, specifically in myocardial infarction progression via regulation of cuproptosis .

What experimental models are most suitable for UBE2D3 research?

The selection of experimental models for UBE2D3 research depends on the specific biological process under investigation. Based on recent literature, researchers have successfully employed:

• In vivo mouse models: Myocardial infarction (MI) mouse models created by left anterior descending (LAD) coronary artery ligation have been effective for studying UBE2D3's role in cardiac pathology .

• Human cell lines: The AC16 human cardiomyocyte cell line has proven valuable for investigating UBE2D3's function in oxygen-glucose deprivation (OGD) conditions that mimic myocardial infarction .

• SILAC-labeled cell cultures: Stable isotope labeling by amino acids in cell culture (SILAC) approaches have been successfully used for quantitative proteomic analysis of UBE2D3 targets .

When selecting models, researchers should consider whether they aim to study tissue-specific effects (requiring animal models) or molecular mechanisms (where cell lines may be sufficient).

What are the most reliable antibodies for detecting UBE2D3 in different applications?

Several validated antibodies have demonstrated reliability in recent UBE2D3 research:

• For Western Blot analysis: Invitrogen PA5-119881 antibody has been successfully used in myocardial tissue analysis and cardiomyocyte studies .

• For immunofluorescence studies: Multiple studies have utilized UBE2D3 antibodies in combination with fluorescent secondary antibodies for localization studies.

When selecting antibodies, researchers should verify:

• Species reactivity (human, mouse, rat)

• Applications validated by the manufacturer (WB, IF, IHC, IP)

• Clonality (monoclonal vs. polyclonal)

• Immunogen sequence used to generate the antibody

It is recommended to validate antibody specificity in your experimental system using positive and negative controls, including UBE2D3 knockdown samples.

What is the recommended protocol for Western Blot detection of UBE2D3?

Based on published methodologies, the following Western Blot protocol has proven effective for UBE2D3 detection:

Sample Preparation:

• Extract total protein from tissue or cells using lysis buffer containing 1% protease inhibitor

• Quantify protein using the BCA method (e.g., Beyotime, #P0012S)

Electrophoresis and Transfer:

• Load 20-30 μg of protein per lane on 10-12% SDS-PAGE gels

• Transfer to PVDF or nitrocellulose membranes

Antibody Incubation:

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

• Incubate with primary UBE2D3 antibody (e.g., Invitrogen PA5-119881) at 1:1000 dilution overnight at 4°C

• Wash membranes 3 times with TBST (5 minutes each)

• Incubate with appropriate HRP-conjugated secondary antibody for 1 hour at room temperature

• Wash membranes 3 times with TBST

Detection:

• Develop using ECL substrate

• Use GAPDH (e.g., Invitrogen, PA1-988) as internal reference protein

For quantification, normalize UBE2D3 band intensity to GAPDH using image analysis software.

How can researchers effectively use UBE2D3 antibodies for immunofluorescence studies?

For optimal immunofluorescence detection of UBE2D3, follow these methodological guidelines:

Fixation and Permeabilization:

• Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes

Blocking and Antibody Incubation:

• Block with 5% BSA in PBS for 1 hour at room temperature

• Incubate with primary UBE2D3 antibody (1:100-1:200 dilution) overnight at 4°C

• Wash 3 times with PBS

• Incubate with fluorophore-conjugated secondary antibody for 1 hour at room temperature

• Wash 3 times with PBS

• Counterstain nuclei with DAPI

Co-localization Studies:
For co-localization with cellular structures, researchers can use phalloidin (green) for cytoskeleton visualization as demonstrated in myocardial tissue studies . This approach allows assessment of UBE2D3 localization relative to cellular architecture.

Image Acquisition:
Use confocal microscopy with appropriate filter settings for the selected fluorophores. Include scale bars (e.g., 100 μm) in all images for proper size reference .

What methods are effective for modulating UBE2D3 expression in experimental systems?

Several approaches have been validated for modulating UBE2D3 expression:

Knockdown Strategies:

• siRNA transfection: Successfully employed to knock down UBE2D3 in AC16 cardiomyocytes to study its role in hypoxic damage and cuproptosis .

• shRNA: For stable knockdown in long-term experiments

Validation Methods:

• qRT-PCR: For mRNA expression analysis

• Western Blot: For protein expression verification

When performing knockdown experiments, it is crucial to:

• Include appropriate negative controls (non-targeting siRNA)

• Validate knockdown efficiency at both mRNA and protein levels

• Consider potential off-target effects

• Optimize transfection conditions for your specific cell type

The effectiveness of knockdown can be assessed through functional assays, such as MTT for cell viability, LDH release for cytotoxicity, and TUNEL staining for apoptosis detection, as demonstrated in cardiomyocyte studies .

How can researchers identify in vivo targets of UBE2D3?

Identifying in vivo targets of UBE2D3 requires sophisticated proteomic approaches. Based on current research, two effective methodologies have been established:

SILAC-Based diGly Proteomics:

• Culture cells in media containing either light or heavy isotope-labeled amino acids

• Deplete UBE2D3 in one population (e.g., using siRNA)

• Combine and digest samples

• Enrich for diGly-modified peptides (signature of ubiquitination)

• Perform LC-MS/MS analysis

• Identify proteins with decreased ubiquitination upon UBE2D3 depletion

Label-Free Quantitative diGly-Proteomics:
This alternative approach does not require SILAC labeling but follows similar enrichment and analysis principles for diGly-modified peptides .

TULIP2 Methodology:
This advanced technique has been used to identify direct UBE2D3 targets, including ribosomal proteins RPS10, RPS20, and the autophagy receptor SQSTM1 .

Researchers should note that these techniques require specialized mass spectrometry equipment and bioinformatics expertise for data analysis.

UBE2D3 has been implicated in multiple levels of protein quality control (PQC) and autophagy regulation. Based on proteomic studies, researchers can investigate these functions through:

Ubiquitinome Profiling:
Analysis of UBE2D3-dependent ubiquitination identifies proteins involved in protein quality control as key targets .

Autophagy Markers Assessment:

• Monitor levels and ubiquitination of autophagy receptor SQSTM1 (p62), a direct target of UBE2D3

• Assess autophagic flux using LC3-I to LC3-II conversion

• Evaluate autophagosome formation through fluorescent microscopy

Protein Level Regulation:
UBE2D3 affects protein levels of specific targets, including CRABP1 and TSPAN8, which can be monitored through Western Blot analysis after UBE2D3 depletion .

When investigating UBE2D3's role in these pathways, researchers should consider its potential cooperation with different E3 ligases, as UBE2D3 functions with multiple E3 partners in vivo.

How should researchers interpret contradictory results in UBE2D3 antibody experiments?

When facing contradictory results in UBE2D3 antibody experiments, consider these systematic approaches:

Antibody Validation:

• Confirm antibody specificity using UBE2D3 knockdown or knockout controls

• Test multiple antibodies targeting different epitopes of UBE2D3

• Verify antibody lot-to-lot consistency

Experimental Variables:

• Cell type or tissue-specific differences: UBE2D3 expression and function may vary across tissues (e.g., single-cell analysis has shown differential UBE2D3 expression across endothelial cells, fibroblasts, macrophages, and stromal cells)

• Experimental conditions: Stress conditions like hypoxia significantly elevate UBE2D3 expression

• Detection methods: Different sensitivities between Western Blot, immunofluorescence, and qRT-PCR

UBE2D Family Cross-Reactivity:
Consider potential cross-reactivity with other UBE2D family members (UBE2D1, UBE2D2, UBE2D4) that share high sequence homology.

Post-Translational Modifications:
UBE2D3 function can be regulated by post-translational modifications, which may affect antibody recognition depending on the epitope.

When reporting contradictory results, clearly document all experimental variables and antibody information to facilitate interpretation and reproducibility.

What controls should be included in UBE2D3 antibody-based experiments?

Proper experimental controls are essential for reliable UBE2D3 research:

Positive Controls:

• Cell lines or tissues known to express UBE2D3 (e.g., AC16 cardiomyocytes)

• Recombinant UBE2D3 protein (for Western Blot)

Negative Controls:

• UBE2D3 knockdown or knockout samples

• IgG isotype control for immunoprecipitation

• Secondary antibody-only controls for immunofluorescence

Loading Controls:

• GAPDH for Western Blot normalization (e.g., Invitrogen, PA1-988)

• Total protein staining (Ponceau S, REVERT)

Treatment Controls:

• For OGD experiments: normal oxygen and glucose conditions

• For MI models: sham-operated animals

Biological Replicates:
Minimum of three independent biological replicates for statistical validation, as demonstrated in published UBE2D3 research .

How can researchers assess the specificity of their UBE2D3 antibody?

Rigorous assessment of UBE2D3 antibody specificity is critical for reliable research outcomes:

Genetic Approaches:

• UBE2D3 knockdown validation: Compare antibody signal between control and UBE2D3-depleted samples (using siRNA or shRNA)

• Overexpression analysis: Confirm increased signal in UBE2D3-overexpressing cells

Biochemical Approaches:

• Peptide competition assay: Pre-incubate antibody with immunizing peptide before detection

• Western Blot analysis: Verify single band at the expected molecular weight (~17 kDa)

• Immunoprecipitation-Mass Spectrometry: Confirm UBE2D3 as the primary protein pulled down

Multiple Detection Methods:
Cross-validate findings using different techniques (Western Blot, immunofluorescence, immunohistochemistry) and different antibodies.

Band Verification:
For Western Blot, compare observed band size with theoretical molecular weight and check against positive control samples.

What emerging techniques show promise for advancing UBE2D3 research?

Emerging methodologies that will likely enhance UBE2D3 research include:

Single-Cell Analysis:
Recent research has utilized single-cell sequencing to identify UBE2D3 expression patterns across different cell types. This approach revealed significant differences in UBE2D3 expression in endothelial cells, fibroblasts, macrophages, and stromal cells, suggesting cell type-specific functions .

Proximity Labeling Approaches:
BioID or TurboID-based proximity labeling systems can identify proteins in close proximity to UBE2D3, helping map its protein interaction network in living cells.

CRISPR-Based Technologies:

• CRISPR knockout/knockin for precise genetic manipulation of UBE2D3

• CRISPRi/CRISPRa for tunable control of UBE2D3 expression

• Base editing for introducing specific mutations to study structure-function relationships

Advanced Proteomics:
Combining UBE2D3 manipulation with techniques like TULIP2 has already identified direct substrates including RPS10, RPS20, and SQSTM1 . Further refinement of these approaches will likely reveal additional targets.

What therapeutic applications might arise from UBE2D3 research?

Current research suggests several potential therapeutic applications based on UBE2D3 function:

Cardiovascular Disease:
UBE2D3 has been shown to promote hypoxic damage of cardiomyocytes by regulating cuproptosis during myocardial infarction. Targeting UBE2D3 might therefore represent a novel strategy for cardioprotection .

Cancer Therapy:
UBE2D3 expression is altered in multiple cancer types, and its levels affect responses to radiation therapy and all-trans retinoic acid (ATRA) treatment . This suggests potential for:

• Biomarker development for treatment response prediction

• Combination therapies targeting UBE2D3 to enhance current treatments

Protein Quality Control Disorders:
Given UBE2D3's role in protein quality control pathways, it may represent a target for neurodegenerative diseases characterized by protein aggregation.

Future drug development focusing on UBE2D3 inhibition could potentially address multiple pathological conditions, though additional research is needed to fully understand tissue-specific effects and potential side effects.