UBE2V2 Antibody

What is UBE2V2 and what is its role in cellular mechanisms?

UBE2V2 (Ubiquitin-conjugating Enzyme E2 Variant 2), also known as MMS2 and UEV2, is a 145 amino acid protein with a predicted molecular weight of approximately 16.5 kDa. It belongs to the ubiquitin-conjugating enzyme (E2) family but lacks an active site cysteine residue, rendering it catalytically inactive on its own .

UBE2V2 primarily functions by forming a catalytically active complex with UBE2N/Ubc13, which specifically catalyzes the formation of Lys63-linked poly-Ubiquitin chains . This complex is essential for genome maintenance in the nucleus through DNA damage repair pathways . Following DNA damage, the UBE2N/Ubc13-UBE2V2 complex forms foci in the nucleus and functions in multiple DNA repair pathways, including:

• Interacting with the SHPRH and HLTF Ubiquitin ligases (E3s) to promote poly-ubiquitination of PCNA, a key step in the postreplication repair pathway

• Functioning with the RNF8 E3 to poly-ubiquitinate Histone H2A and Histone H2AX near DNA double-stranded breaks, facilitating the recruitment of DNA repair effector proteins

Research has shown that UBE2V2 demonstrates significant tumorigenicity in many cancers, with overexpression linked to tumor progression and metastasis .

How can researchers differentiate UBE2V2 from other ubiquitin-conjugating enzymes in experiments?

Differentiating UBE2V2 from other ubiquitin-conjugating enzymes requires a multi-faceted approach:

• Antibody specificity validation: Use recombinant fusion proteins containing amino acids 1-145 of human UBE2V2 (NP_003341.1) as a positive control . This specific sequence (MAVSTGVKVPRNFRLLEELEEGQKGVGDGTVSWGLEDDEDMTLTRWTGMIIGPPRTNYEN RIYSLKVECGPKYPEAPPSVRF VTKINMNGINNSSGMVDARSIPVLAKWQNSYSIKVVLQELRRLMMSKENMKLPQPPEGQTYNN) differs from other E2 enzymes .

• Functional assays: Unlike classic E2 enzymes, UBE2V2 forms a heterodimer with UBC13, catalyzing K63-linked non-canonical polyubiquitin chains. Co-immunoprecipitation with UBC13 can confirm UBE2V2 identity .

• Expression pattern analysis: UBE2V2 shows distinct expression patterns across tissues. In pathological contexts like lung adenocarcinoma, UBE2V2 exhibits characteristic correlations with clinicopathological factors including gender (p = 0.043), stage (p = 0.042), and lymph node metastasis (p = 0.002) .

What are the validated applications for UBE2V2 antibodies and their recommended protocols?

UBE2V2 antibodies have been validated for several experimental applications:

Western Blotting (WB):

• Recommended dilution: 1:500 to 1:2000

• Protocol highlights:

  • Separate proteins using SDS-PAGE gel at 120V

  • Transfer to PVDF membrane at 200mA

  • Block with 5% skim milk at 25°C for 2h

  • Incubate with primary UBE2V2 antibody at 4°C for 12h

  • Wash with TBST 3x (20 min each)

  • Incubate with HRP-conjugated secondary antibody (1:10000) at 25°C for 2h

  • Visualize using ECL detection system

Immunohistochemistry (IHC-P):

• Recommended dilution: 1:50 to 1:200

• Protocol highlights:

  • Fix tissue samples with 10% formalin before embedding

  • Cut 4μm-thick paraffin sections

  • Dewax using graded ethanol concentrations

  • Block endogenous peroxidase with 3% H₂O₂

  • Incubate with anti-UBE2V2 antibody (1:100) at 25°C for 2h

  • Wash with PBS 3x (10 min each)

  • Apply secondary antibody at 25°C for 1h

  • Visualize with DAB solution

  • Counterstain with hematoxylin

ELISA:

• Validated for sandwich ELISA techniques

• Shows higher specificity compared to conventional competitive ELISA kits

How should researchers evaluate and score UBE2V2 expression in immunohistochemistry studies?

For consistent and reproducible evaluation of UBE2V2 expression in IHC studies, researchers should implement the following scoring system:

Proportion Score (percentage of stained cells):

• Score 1: 1%-30% positive cells

• Score 2: 31%-50% positive cells

• Score 3: 51%-70% positive cells

• Score 4: 71%-100% positive cells

Intensity Score (staining intensity):

• Score 0: No staining

• Score 1: Light yellow

• Score 2: Deep yellow

• Score 3: Brown particles

Final Expression Score (product of proportion and intensity scores):

• Low expression: 1-4 points

• High expression: 6-12 points

For accurate assessment, three experienced pathologists should independently evaluate the staining results. This methodology has been validated in studies examining UBE2V2's relationship with clinicopathological parameters in lung adenocarcinoma .

What is the significance of UBE2V2 in lung adenocarcinoma progression and prognosis?

UBE2V2 has emerged as a significant biomarker and potential therapeutic target in lung adenocarcinoma (LUAD):

Prognostic Value:

• UBE2V2 serves as an independent prognostic indicator for LUAD patients based on:

  • TCGA prediction (HR: 1.497, p = 0.012)

  • Immunohistochemistry validation (HR: 1.864, p = 0.044)

• High UBE2V2 expression correlates with poor survival outcomes in LUAD patients

Clinicopathological Correlations:

• UBE2V2 expression significantly correlates with:

  • Gender (p = 0.043)

  • Tumor stage (p = 0.042)

  • Lymph node metastasis (p = 0.002)

  • T classification in TCGA data (p = 0.013)

Cellular Mechanisms:

• Knockdown of UBE2V2 in LUAD cells results in:

  • Reduced migration capacity

  • Cell cycle arrest in G1 phase

  • Increased apoptosis

  • Decreased proliferation

  • Downregulation of PCNA

  • Upregulation of P53 and ƳH2AX

These findings demonstrate that UBE2V2 promotes LUAD progression through multiple cellular mechanisms, making it a promising biomarker for prognosis assessment and a potential therapeutic target for LUAD treatment.

How does UBE2V2 influence the epithelial-mesenchymal transition (EMT) and metastasis in cancer cells?

UBE2V2 plays a critical role in regulating epithelial-mesenchymal transition (EMT) and promoting metastasis in cancer cells:

EMT Regulation Mechanism:
UBE2V2 knockdown experiments in LUAD cell lines (A549 and SPCA1) revealed significant changes in EMT-related proteins:

Metastasis Impact:

• Transwell assays demonstrated that UBE2V2 knockdown significantly inhibited the migration ability of LUAD cells

• The correlation between UBE2V2 expression and lymph node metastasis (p = 0.002) observed in IHC studies supports its role in promoting cancer cell dissemination

• This effect has been observed in multiple cancer types, including melanoma where inhibiting UBE2V2 expression upregulated E-cadherin

These findings suggest that UBE2V2 promotes metastasis by modulating EMT-related proteins, specifically by suppressing epithelial characteristics (E-cadherin) while enhancing mesenchymal properties (N-cadherin, vimentin) and invasion capacity (MMP2). This mechanism represents a potential therapeutic target for preventing cancer metastasis.

What is the relationship between UBE2V2 expression and immune cell infiltration in tumors?

UBE2V2 demonstrates significant associations with immune cell infiltration in tumor microenvironments, particularly in lung adenocarcinoma:

Correlations with Immune Cell Types:
Analysis through the TIMER database revealed that UBE2V2 expression is:

Immunotherapy Implications:

• UBE2V2 mRNA levels positively correlate with PD-L1 mRNA levels in LUAD

• Immunohistochemistry confirmed a positive correlation between UBE2V2 protein levels and PD-L1 expression in clinical samples

• UBE2V2 expression is negatively correlated with type II interferon response, suggesting immunosuppressive effects

These correlations suggest that UBE2V2 may influence the tumor microenvironment by modulating immune cell infiltration and potentially contributing to immune evasion through its relationship with PD-L1. This understanding could inform immunotherapy strategies for cancer treatment, particularly in tumors with high UBE2V2 expression.

What techniques can researchers use to investigate UBE2V2's role in DNA damage repair pathways?

Researchers can employ several sophisticated techniques to elucidate UBE2V2's function in DNA damage repair:

Foci Formation Assays:

• Immunofluorescence microscopy to visualize UBE2V2 and UBE2N/Ubc13 nuclear foci formation following DNA damage

• Co-localization studies with DNA damage markers (γH2AX) using confocal microscopy

• Live-cell imaging with fluorescently tagged UBE2V2 to track recruitment kinetics to damage sites

Protein-Protein Interaction Studies:

• Co-immunoprecipitation to isolate UBE2V2 complexes with UBE2N/Ubc13, SHPRH, HLTF, and RNF8

• Proximity ligation assays to visualize in situ interactions between UBE2V2 and partner proteins

• FRET/BRET assays to measure direct interactions in living cells

Ubiquitination Analysis:

• In vitro ubiquitination assays to measure UBE2V2-UBE2N/Ubc13 catalytic activity

• Immunoblotting for K63-linked ubiquitin chains on target proteins (PCNA, H2A, H2AX)

• Mass spectrometry to identify ubiquitination sites and ubiquitin chain topology

Functional DNA Repair Assays:

• Comet assays to measure DNA strand break repair efficiency

• HR and NHEJ reporter assays to assess repair pathway choice

• Clonogenic survival assays following DNA damage in UBE2V2-depleted cells

Chromatin Association:

• Chromatin immunoprecipitation (ChIP) to detect UBE2V2 recruitment to damage sites

• Chromatin fractionation to assess UBE2V2 association with chromatin following damage

• Sequential ChIP to analyze co-occupancy with other repair factors

These methodologies, when used in combination, provide comprehensive insights into UBE2V2's mechanistic role in DNA damage response pathways.

How can researchers effectively knockdown UBE2V2 to study its loss-of-function effects in cancer models?

Researchers can employ multiple approaches to achieve effective UBE2V2 knockdown for loss-of-function studies:

RNA Interference (RNAi) Strategies:

• shRNA approach: Validated shRNA constructs (particularly shRNA-2) have demonstrated high knockdown efficiency in LUAD cell lines

• siRNA alternatives: For transient knockdown experiments when stable modification is not required

Design considerations:

• Target specificity: Ensure sequences specifically target UBE2V2 without affecting homologous proteins (like UBE2V1)

• Efficiency validation: Quantify knockdown at both mRNA level (RT-qPCR) and protein level (Western blot)

• Multiple constructs: Test multiple shRNA sequences as knockdown efficiency varies (e.g., shRNA-2 showed superior knockdown compared to other constructs)

CRISPR-Cas9 Gene Editing:

• More complete gene inactivation compared to RNAi

• Design guide RNAs targeting early exons of UBE2V2

• Validate knockout through sequencing and Western blotting

Validation Methodologies:

• Western blot protocol: Use anti-UBE2V2 antibody at 1:500-1:2000 dilution with appropriate positive control samples (SW480, SKOV3, HeLa, HL-60)

• RT-qPCR: Design primers spanning exon junctions to avoid genomic DNA amplification

• Functional validation: Confirm expected phenotypes (decreased migration, increased apoptosis, cell cycle arrest)

Experimental Controls:

• Include non-targeting shRNA controls (shCon) processed identically to experimental samples

• Consider rescue experiments with shRNA-resistant UBE2V2 to confirm specificity

• Include wild-type cells as additional controls

By following these methodological approaches, researchers can effectively achieve UBE2V2 knockdown to study its functional role in cancer progression and potential as a therapeutic target.

What are common challenges in UBE2V2 antibody-based experiments and how can they be resolved?

Researchers working with UBE2V2 antibodies may encounter several technical challenges that can be addressed with specific solutions:

Western Blot Issues:

Immunohistochemistry Challenges:

Cell Line Selection Issues:

• Variable UBE2V2 expression across cell lines can affect experimental outcomes

• Solution: Screen potential lines by Western blot; A549 and SPCA1 cell lines are validated models for UBE2V2 studies in LUAD

Knockdown Efficiency Concerns:

• Different shRNA constructs show variable knockdown efficiency

• Solution: Test multiple shRNA sequences; shRNA-2 shows superior knockdown compared to other constructs in published studies

Implementing these troubleshooting strategies can significantly improve the reliability and reproducibility of UBE2V2 antibody-based experiments.

How should researchers interpret contradictory results when studying UBE2V2 across different cancer types?

When facing contradictory results in UBE2V2 studies across cancer types, researchers should employ a systematic interpretive framework:

Context-Dependent Expression Analysis:

• UBE2V2 shows tissue-specific expression patterns that affect its function

• Recommendation: Perform comprehensive profiling across multiple tissue types using standardized methodologies

• Observation: UBE2V2 is highly expressed in malignant melanoma , prostate cancer , and lung adenocarcinoma , but functional effects may differ

Molecular Interaction Networks:

• UBE2V2's function depends on interaction partners that vary by cancer type

• Approach: Map protein-protein interactions using co-immunoprecipitation followed by mass spectrometry

• Example: UBE2V2 co-expression analysis reveals different correlation patterns across cancers (Figure S1A and Table S1)

Methodological Reconciliation:

Statistical Consideration:

• Different statistical thresholds and sample sizes affect significance determination

• Solution: Standardize significance thresholds (p<0.05) and use adequate sample sizes (n=91 in validated LUAD studies)

• Apply multivariate Cox analysis to control for confounding variables when determining prognostic value

By applying this systematic framework, researchers can meaningfully interpret seemingly contradictory results across cancer types and develop a more nuanced understanding of UBE2V2's context-dependent roles in oncogenesis.

What emerging techniques might enhance the study of UBE2V2 beyond current antibody-based methods?

Several cutting-edge technologies are poised to advance UBE2V2 research beyond traditional antibody-based approaches:

Proximity-Based Proteomic Technologies:

• BioID and TurboID for in vivo identification of UBE2V2 interaction partners

• APEX2-based proximity labeling to map UBE2V2's spatial interactome within specific subcellular compartments

• These techniques overcome limitations of antibody-dependent co-immunoprecipitation and can detect transient interactions

CRISPR-Based Functional Genomics:

• CRISPR activation/interference (CRISPRa/CRISPRi) for precise modulation of UBE2V2 expression

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

• CRISPR screens to identify synthetic lethal interactions with UBE2V2 in cancer contexts

Advanced Imaging Techniques:

• Super-resolution microscopy (STORM/PALM) for visualizing UBE2V2 localization at nanometer resolution

• Lattice light-sheet microscopy for real-time tracking of UBE2V2 dynamics during DNA damage response

• Correlative light and electron microscopy (CLEM) to visualize UBE2V2 in the context of cellular ultrastructure

Structural Biology Approaches:

• Cryo-electron microscopy to determine UBE2V2-UBE2N complex structures at near-atomic resolution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to study conformational dynamics

• AlphaFold2 and other AI-based structural prediction tools to model UBE2V2 interactions

Single-Cell Technologies:

• Single-cell proteomics to analyze UBE2V2 expression heterogeneity in tumors

• Spatial transcriptomics to map UBE2V2 expression patterns within the tumor microenvironment

• CITE-seq to simultaneously profile UBE2V2 protein levels and gene expression in single cells

These emerging technologies will enable more precise characterization of UBE2V2's functions, interactions, and dynamics in normal and pathological contexts, potentially revealing new therapeutic opportunities.

What therapeutic strategies targeting UBE2V2 show promise for cancer treatment?

Based on current research findings, several therapeutic strategies targeting UBE2V2 show potential for cancer treatment:

Direct UBE2V2 Inhibition Approaches:

• Small molecule inhibitors disrupting UBE2V2-UBE2N interaction

• Peptide-based inhibitors targeting the UBE2V2 binding interface

• Rationale: Knockdown studies demonstrate that UBE2V2 inhibition reduces cancer cell proliferation and increases apoptosis in LUAD models

Combination Therapy Opportunities:

RNA-Based Therapeutics:

• miRNA delivery: miR-499a inhibits prostate cancer cell proliferation by targeting UBE2V2

• siRNA/shRNA approaches: Validated constructs (e.g., shRNA-2) demonstrate efficacy in preclinical models

• Antisense oligonucleotides targeting UBE2V2 mRNA

Biomarker-Guided Patient Selection:

• UBE2V2 expression can identify patients likely to benefit from targeted therapies

• Gender-specific approaches may be warranted (UBE2V2 expression correlates with gender, p=0.043)

• Lymph node metastasis status (p=0.002) could guide adjuvant therapy decisions

Immune Modulation Strategies:

• Targeting UBE2V2 may enhance immune infiltration by B cells, CD4+ T cells, macrophages, and dendritic cells

• Combined UBE2V2 inhibition with immune checkpoint blockade could overcome immune evasion mechanisms