UBE2H Antibody

What is UBE2H and why is it significant in research?

UBE2H (Ubiquitin-conjugating enzyme E2H) is a critical component of the ubiquitin-proteasome system that regulates protein degradation and maintains cellular homeostasis. This 183 amino acid protein (21 kDa) facilitates the covalent attachment of ubiquitin to target proteins, tagging them for degradation or altering their cellular location and function. UBE2H is particularly important for histone H2A degradation, influencing gene expression and chromatin dynamics. Its high conservation across species (100% identity to mouse homolog and 98% to frog and zebrafish homologs) underscores its fundamental role in cellular processes . Recent research has revealed its significance in erythropoiesis and potential implications in cancer progression, particularly lung adenocarcinoma .

What are the common aliases for UBE2H in scientific literature?

When reviewing literature, researchers should be aware of multiple nomenclatures for UBE2H:

• E2 ubiquitin-conjugating enzyme H

• UBC8 homolog

• UBCH2

• Ubiquitin carrier protein H

• E2-20K

• GID3

• UBCH

• Ubiquitin-protein ligase H

This diversity in naming can complicate literature searches, so comprehensive search strategies should include all known aliases.

What are the typical molecular characteristics of UBE2H that affect antibody selection?

UBE2H is characterized by:

• Protein size: 183 amino acids

• Molecular weight: 21 kDa (observed in Western blots)

• Multiple isoforms from alternative splicing

• High cross-species conservation

• Expression in multiple tissues including brain, kidney, and various cell lines

When selecting antibodies, researchers should consider the specific isoform targeted, epitope location, and cross-reactivity with homologs from other species if conducting comparative studies.

Which experimental applications have been validated for UBE2H antibodies?

Based on current literature and commercial antibody validation data, UBE2H antibodies have been successfully employed in:

Researchers should note that optimal dilutions are sample-dependent and should be determined experimentally for each new study context .

How should researchers optimize Western blot protocols for UBE2H detection?

For optimal Western blot detection of UBE2H:

• Sample preparation: Use RIPA or similar lysis buffers with protease inhibitors

• Loading control: Include GAPDH or β-actin as housekeeping proteins

• Gel percentage: 12-15% SDS-PAGE gels are optimal for resolving this 21 kDa protein

• Transfer conditions: Semi-dry or wet transfer at 100V for 60-90 minutes

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Start with 1:1000 dilution, incubate overnight at 4°C

• Secondary antibody: HRP-conjugated at 1:5000-1:10000, 1 hour at room temperature

• Special considerations: For studying UBE2H regulation, include proteasome inhibitors (e.g., MG132) in experimental designs to distinguish between stability effects and differentiation effects

These parameters may require optimization based on specific experimental conditions and antibody sources.

What are the key considerations for using UBE2H antibodies in immunohistochemistry?

For successful IHC detection of UBE2H:

• Tissue preparation: Formalin-fixed, paraffin-embedded (FFPE) sections (4-6 μm)

• Antigen retrieval: Recommended with TE buffer pH 9.0; alternative: citrate buffer pH 6.0

• Blocking: 10% normal serum from secondary antibody species, 1 hour at room temperature

• Primary antibody dilution: Start with 1:100, optimize as needed (range: 1:50-1:500)

• Incubation: Overnight at 4°C

• Detection system: Appropriate secondary antibody with HRP/DAB detection

• Positive controls: Colon tissue has shown reliable positivity for UBE2H

• Known expression patterns: Nuclear and cytoplasmic localization expected

Signal specificity should be confirmed using appropriate negative controls and validation methods.

How does UBE2H expression change during erythroid differentiation?

Recent research demonstrates dynamic regulation of UBE2H during erythropoiesis:

• Expression profile: UBE2H protein levels increase significantly during terminal erythroid differentiation

• Correlation: UBE2H upregulation parallels the induction of erythroid-specific proteins like CD235a (glycophorin A) and hemoglobin

• Comparative analysis: UBE2H belongs to a cluster of six E2 enzymes that progressively accumulate until day 12 of differentiation, including UBE2O, a known mediator of ribosomal clearance in reticulocytes

• Regulation: UBE2H is transcriptionally regulated by the essential erythroid nuclear protein TAL1

• Model systems: This pattern has been confirmed in both HUDEP2 and CD34+ cell differentiation models

These findings suggest that UBE2H plays a specific temporal role in erythrocyte development that merits further investigation.

What functional phenotypes emerge from UBE2H depletion in erythroid cells?

CRISPR-Cas9-mediated inactivation of UBE2H in erythroid progenitors reveals several phenotypic consequences:

• Accelerated differentiation: UBE2H-deficient cells show spontaneous and accelerated erythroid maturation

• Enucleation defects: Cells show inefficient enucleation, a critical step in terminal erythropoiesis

• Molecular changes: Altered expression of developmental stage-specific proteins

• Mechanistic implications: Suggests UBE2H functions as a regulator of orderly erythroid maturation progression

• Research questions: Additional assays comparing viability and functionality of control versus UBE2H-deficient orthochromatic erythroblasts would provide further insights

These findings establish UBE2H as a critical regulator of proper timing in erythroid development.

What is the relationship between UBE2H and the CTLH E3 ligase complex in erythroid cells?

UBE2H functions as part of a regulatory module with the CTLH E3 ubiquitin ligase complex:

• Interaction specificity: UBE2H is the preferred E2 enzyme for the CTLH E3 ubiquitin ligase

• Parallel expression: UBE2H and most CTLH complex subunits show similar upregulation during erythroid maturation

• Complex dynamics: CTLH complexes undergo stage-specific modulation of composition during differentiation

• Subunit switching: RANBP9 and RANBP10 homologs show inverse expression patterns during differentiation

• Complex assembly: Sucrose density gradient analysis confirms that CTLH components sediment at ≥670 kDa, consistent with supramolecular assemblies

• Function: UBE2H-CTLH modules appear to control the orderly progression of human erythropoiesis

This evidence establishes a functional E2-E3 module with stage-specific regulation during erythroid development.

What evidence links UBE2H to lung adenocarcinoma (LUAD) progression?

Multiple lines of evidence implicate UBE2H in LUAD progression:

• Differential expression: RNA sequencing revealed higher UBE2H expression in tumor tissue compared to normal tissue

• Metastatic correlation: Highest expression of UBE2H was observed in malignant pleural tumors compared to primary tumors

• Staging correlation: UBE2H expression progressively increases with LUAD stage, with statistically significant differences between normal vs. all tumor stages (p-values ranging from 1.6×10^-4 to 2.4×10^-11)

• Survival impact: High UBE2H expression correlates with poor survival in multiple independent datasets

• Hypoxia connection: UBE2H shows positive correlation with hypoxia-related genes (AP2B1, BNIP3L, ENOSF1, GPR31, HNRNPC, KCNMA1, KPNB1, LGALS1, LOX, NCL, NDRG1, RPS3)

These findings suggest UBE2H could serve as both a prognostic biomarker and potential therapeutic target in LUAD.

How does UBE2H influence cellular migration and metastasis in cancer models?

Functional studies reveal UBE2H's role in cancer cell migration and metastasis:

• Migration capacity: UBE2H knockdown significantly inhibits migration in wound-healing and transwell migration assays

• EMT regulation: UBE2H suppression reverses epithelial-mesenchymal transition (EMT) signaling pathways

• Molecular changes: UBE2H knockdown reduces mesenchymal phenotype-associated molecules including N-cadherin and Snail

• Differential effects: While N-cadherin and Snail are downregulated, Vimentin expression remains unchanged

• Regulatory implications: Suggests UBE2H selectively regulates specific EMT effectors to promote metastatic capacity

These mechanisms potentially explain UBE2H's association with metastasis and poor outcomes in lung cancer.

What regulatory mechanisms control UBE2H expression in cancer contexts?

Multiple regulatory mechanisms appear to control UBE2H expression in cancer:

• MicroRNA regulation: Five microRNAs (miR-101, miR-30a, miR-30b, miR-328, miR-497) predicted to target UBE2H are associated with favorable prognosis

• Copy number variation: UBE2H expression shows significant correlation with copy number variation in LUAD samples

• Promoter methylation: Correlation between UBE2H expression and methylation status of the UBE2H promoter has been investigated

• Hypoxic regulation: UBE2H expression correlates with hypoxia-related genes, suggesting potential regulation through hypoxia pathways

• Post-transcriptional control: Proteasome inhibition with MG132 affects UBE2H levels, indicating regulation at the protein stability level

Understanding these regulatory mechanisms could inform therapeutic strategies targeting UBE2H in cancer contexts.

How might differential CTLH complex composition affect UBE2H function?

Emerging evidence suggests functional heterogeneity in CTLH-UBE2H interactions:

• Subunit switching: RANBP9 and RANBP10 show inverse expression patterns during erythroid differentiation

• Complex modulation: Sucrose density gradient analysis confirms stage-specific changes in CTLH complex composition

• Research gap: Current data don't fully resolve whether RANBP9 and RANBP10 complexes assemble additional subunits

• Methodological approach: Immunoprecipitation studies using specific antibodies or nanobodies for complex components (like ARMC8) can further elucidate complex composition

• Future directions: Comparative substrate identification for different CTLH complex compositions could reveal functional specialization

Investigating these complex dynamics represents an important frontier in understanding UBE2H biology.

What is the mechanism by which MAEA stabilizes UBE2H expression?

The interdependence between MAEA (CTLH complex component) and UBE2H requires further investigation:

• Current knowledge: Re-expression of MAEA with mutations in ubiquitylation sites fails to restore UBE2H expression

• Mechanistic hypothesis: CTLH complex activity may be required to maintain UBE2H expression

• Research limitations: Current evidence is based on single transfection experiments that require quantitative validation

• Interdependence: MAEA and RMND5A expression are interdependent, necessitating control experiments ensuring RMND5A restoration

• Alternative mechanism: UBE2H levels may be regulated through ubiquitin modification itself, similar to other E2s (UBE2T, UBE2E)

• Experimental approach: Investigating whether proteasome inhibition with MG132 affects UBE2H through stabilization or via effects on differentiation requires analysis of additional differentiation markers

These questions highlight the complexity of UBE2H regulation within ubiquitin pathway networks.

What technical considerations are critical when studying UBE2H autoregulation via ubiquitination?

Investigation of UBE2H regulation through the ubiquitin system requires specific technical approaches:

• Inhibitor approaches: Proteasome inhibitors (MG132) can distinguish between stability-dependent and differentiation-dependent effects

• Ubiquitination detection: Use of ubiquitin mutants (K48R, K63R) can help identify specific ubiquitin chain topologies

• Internal controls: Always monitor additional differentiation markers to separate direct effects on UBE2H from indirect effects via differentiation

• Mutation studies: Targeted mutagenesis of putative ubiquitination sites on UBE2H can identify regulatory residues

• Experimental model validation: Ensure experimental models adequately recapitulate the physiological context where regulation occurs

• Quantitative validation: Implement multiple experimental replicates with appropriate statistical analysis to validate findings

• Complementary approaches: Combine biochemical techniques with genetic approaches (CRISPR-Cas9) to validate mechanisms

How should researchers address cross-reactivity concerns with UBE2H antibodies?

To ensure specificity when using UBE2H antibodies:

• Validate with appropriate controls:

  • Positive controls: HEK-293 cells, mouse/rat brain and kidney tissues, HeLa cells

  • Negative controls: UBE2H knockout or knockdown samples

  • Blocking peptide competition

• Consider cross-reactivity with homologous proteins:

  • UBE2H has high sequence similarity with other E2 enzymes

  • Verify antibody specificity against recombinant UBE2H and related proteins

• Validate across multiple applications:

  • An antibody performing well in Western blot may not work in IHC

  • Use complementary detection methods to confirm findings

• When using nanobodies:

  • Specify whether targeting ARMC8 α or β isoforms

  • Include appropriate negative controls beyond "mock" controls

Careful antibody validation is critical for ensuring the reliability and reproducibility of UBE2H research.

What approaches can resolve conflicting findings in UBE2H functional studies?

When faced with inconsistent results in UBE2H research:

• Consider cell type and context dependency:

  • UBE2H may function differently in erythroid cells versus cancer cells

  • Microenvironment factors like hypoxia may alter UBE2H function

• Examine technical variables:

  • Antibody specificity and detection methods

  • Cell culture conditions and passage number

  • Knockdown/knockout efficiency and compensation mechanisms

• Assess experimental timelines:

  • Acute versus chronic UBE2H depletion may yield different phenotypes

  • Stage-specific effects during differentiation processes

• Evaluate compensatory mechanisms:

  • Other E2 enzymes may compensate for UBE2H loss

  • Alternative pathways may be activated in different contexts

• Implement diverse methodological approaches:

  • Combine genetic, biochemical, and cellular techniques

  • Use multiple independent cell models and primary samples

This multi-faceted approach can help reconcile seemingly contradictory findings about UBE2H function.