UBE2O Antibody

What is UBE2O and what cellular functions does it regulate?

UBE2O, also known as KIAA1734 or E2-230K, is a 1,292 amino acid protein belonging to the ubiquitin-conjugating enzyme family. It catalyzes the ATP-dependent covalent attachment of ubiquitin to specific target proteins, marking them for proteasomal degradation . UBE2O is predominantly expressed in heart and skeletal muscle tissues and plays critical roles in:

• Protein degradation and homeostasis maintenance

• Cell cycle regulation and apoptosis

• Circadian rhythm regulation (via BMAL1 interaction)

• Interferon signaling pathway modulation

• Cancer progression through various mechanisms

UBE2O has a calculated molecular mass of 141 kDa, though in experimental settings it can be detected at varying molecular weights between 140-170 kDa and 200-230 kDa . The gene encoding UBE2O is located on human chromosome 17, a region with high gene density that also contains important tumor suppressor genes like p53 and BRCA1 .

What applications are UBE2O antibodies validated for?

UBE2O antibodies have been validated for multiple research applications as shown in the table below:

Positive Western blot detection has been confirmed in multiple cell lines including HeLa, A431, and Jurkat cells, while positive IHC has been observed in human heart tissue . The antibody should be titrated in each testing system to obtain optimal results as reactivity can be sample-dependent .

What is the molecular weight of UBE2O and what bands should I expect in Western blots?

While UBE2O has a calculated molecular weight of 141 kDa (1292 amino acids), experimental observations reveal more complex banding patterns:

• The main observed molecular weight is approximately 150 kDa

• Additional bands are frequently observed at 200-230 kDa

• These variations have been documented in multiple published studies (PMID: 28774900, 23455153, 28774922, 11311559)

The variability in observed molecular weights may result from post-translational modifications, alternative splicing, or protein complex formation. When conducting Western blots, it is advisable to include positive control lysates from cells known to express UBE2O, such as HeLa or Jurkat cells, to properly identify specific bands .

How does UBE2O regulate circadian rhythms through BMAL1 interaction?

UBE2O has been identified as a regulator of the cellular clock through its interaction with the BMAL1 protein (Aryl hydrocarbon receptor nuclear translocator-like protein 1), a core component of the circadian clock machinery . This regulation occurs through the following mechanisms:

• Direct protein-protein interaction between UBE2O and BMAL1, confirmed through immunoprecipitation experiments in both overexpression systems and endogenous contexts

• UBE2O mediates the ubiquitination of BMAL1, targeting it for proteasomal degradation

• This interaction affects BMAL1 protein levels and consequently impacts its transcriptional activity

The interaction between UBE2O and BMAL1 has been validated in multiple experimental systems, including HEK293T cells with overexpressed proteins and through endogenous co-immunoprecipitation in both mouse neuroblastoma N2a cells and mouse brain lysates . Researchers studying circadian rhythm mechanisms should consider examining UBE2O expression and activity when investigating alterations in BMAL1 function, as this regulatory pathway represents an important post-translational control mechanism for circadian rhythm homeostasis.

What is UBE2O's role in interferon-α signaling and its potential therapeutic implications?

UBE2O has emerged as a significant regulator of interferon-α efficacy through its targeting of IFIT3 (interferon-induced protein with tetratricopeptide repeats 3), a mediator of interferon signaling . Key research findings include:

• UBE2O negatively regulates interferon-α/β signaling by promoting the ubiquitination and degradation of IFIT3

• Knockdown of UBE2O significantly enhances interferon-α effectiveness in cancer cells

• K236 has been identified as a critical ubiquitination site in IFIT3

• UBE2O inhibition improves interferon-α efficacy in both in vitro and in vivo experiments

This relationship has significant therapeutic implications, particularly for hepatocellular carcinoma (HCC) treatment, where interferon resistance poses a major challenge. Interestingly, arsenic trioxide (ATO) treatment inhibits UBE2O activity and increases IFIT3 expression, thereby enhancing interferon-α effectiveness . Researchers investigating interferon resistance mechanisms should consider UBE2O as a potential therapeutic target, especially in combination therapies aimed at enhancing interferon efficacy.

How can I effectively validate UBE2O knockdown or overexpression in experimental models?

Proper validation of UBE2O manipulation in experimental models is crucial for ensuring reliable results. Based on published methodologies, we recommend the following approach:

For UBE2O knockdown validation:

• Western blot analysis using specific anti-UBE2O antibodies (1:500-1:1000 dilution) to confirm protein reduction

• qRT-PCR to verify decreased mRNA expression

• Functional assays to demonstrate expected phenotypic changes (e.g., altered interferon sensitivity or circadian gene expression)

For UBE2O overexpression validation:

• Western blot to confirm increased protein expression

• Immunofluorescence to visualize subcellular localization (particularly important as UBE2O function may vary by cellular compartment)

• Co-immunoprecipitation experiments to verify interaction with known substrates

When establishing stable cell lines with modified UBE2O expression, regularly confirm the altered expression levels throughout extended experiments, as compensatory mechanisms may emerge over time . Additionally, include multiple control cell lines and biological replicates to account for clonal variation.

What are the optimal protocols for immunoprecipitation using UBE2O antibodies?

Based on published research methodologies, the following protocol outline is recommended for UBE2O immunoprecipitation experiments:

• Cell/tissue lysate preparation:

  • Lyse cells in a buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, with freshly added protease inhibitors

  • Include 10 mM N-ethylmaleimide (NEM) if studying ubiquitination

  • Clarify lysates by centrifugation at 13,000 rpm for 15 minutes at 4°C

• Immunoprecipitation procedure:

  • Pre-clear lysates with protein A/G beads for 1 hour at 4°C

  • Incubate with anti-UBE2O antibody (2-5 μg per 1 mg of total protein) overnight at 4°C with gentle rotation

  • Add protein A/G beads and incubate for an additional 2-4 hours

  • Wash complexes thoroughly (at least 4-5 times) with lysis buffer

  • Elute proteins by boiling in SDS sample buffer

• For reciprocal verification:

  • Perform reverse immunoprecipitation with antibodies against suspected interacting partners (e.g., BMAL1)

  • Include appropriate negative controls (isotype-matched IgG or unrelated antibody)

This approach has been successfully used to demonstrate the interaction between UBE2O and BMAL1 in both overexpression systems and with endogenous proteins from cell lines and brain tissue .

What controls should be included in UBE2O antibody experiments?

Proper controls are essential for ensuring the reliability and reproducibility of UBE2O antibody experiments. Based on research best practices, the following controls should be incorporated:

For Western blot:

• Positive control: Lysates from cells known to express UBE2O (HeLa, A431, Jurkat cells)

• Negative control: Lysates from UBE2O knockout or knockdown cells

• Loading control: Housekeeping proteins (e.g., GAPDH, β-actin) to normalize protein loading

• Molecular weight marker: To accurately identify UBE2O bands (expected at ~150 kDa and 200-230 kDa)

For immunohistochemistry:

• Positive tissue control: Human heart tissue has shown reliable UBE2O expression

• Negative control: Primary antibody omission or substitution with isotype-matched IgG

• Antigen competition: Pre-incubation of antibody with immunizing peptide to confirm specificity

For immunoprecipitation:

• Input sample: To verify the presence of target proteins before IP

• IgG control: Isotype-matched IgG to identify non-specific interactions

• Reverse IP: Immunoprecipitation with antibodies against interaction partners

For functional studies:

• Multiple UBE2O targeting constructs to rule out off-target effects

• Rescue experiments with wild-type UBE2O to confirm specificity of observed phenotypes

Implementing these controls will significantly enhance the validity and interpretability of UBE2O research findings.

How can I assess the impact of UBE2O on interferon-α efficacy in experimental models?

To evaluate the influence of UBE2O on interferon-α effectiveness, researchers can implement the following experimental approaches based on published methodologies:

• In vitro cell-based assays:

  • Colony formation assay: Treat cells with interferon-α (1000–6000 U/ml) for 48-72 hours after UBE2O manipulation, then seed 1000 cells in 6-well plates and culture for 14 days before crystal violet staining and colony counting

  • Wound healing assay: Assess cell migration capacity following interferon-α treatment in UBE2O-manipulated versus control cells

  • Cell viability/proliferation assays: Determine dose-response curves to interferon-α treatment with UBE2O knockdown or overexpression

• Molecular analysis:

  • Western blot analysis of IFIT3 expression and stability (a direct substrate of UBE2O)

  • Analysis of interferon signaling pathway components (JAK-STAT activation, ISG expression)

  • Ubiquitination assays to detect changes in IFIT3 ubiquitination status

• In vivo xenograft models:

  • Establish subcutaneous xenografts using cells with stable UBE2O knockdown or control cells

  • Administer interferon-α (5 × 10^6 U/kg daily) or saline control

  • Monitor tumor growth and analyze molecular markers in tumor tissue

• Pharmacological approach:

  • Test UBE2O inhibitors (e.g., arsenic trioxide at 2.5-5 mg/kg) alone or in combination with interferon-α

  • Analyze tumor growth, IFIT3 expression, and interferon signaling pathway activation

These methodologies provide a comprehensive framework for investigating UBE2O's impact on interferon signaling and potential therapeutic applications.

What are the key considerations for studying UBE2O in different tissue contexts?

UBE2O expression and function vary significantly across tissue types, necessitating tailored experimental approaches. Consider the following tissue-specific factors:

• Expression profile considerations:

  • UBE2O is predominantly expressed in heart and skeletal muscle

  • Expression levels should be verified in the specific tissue/cell type under investigation

  • Antibody validation is crucial as detection sensitivity may vary by tissue type

• Tissue-specific interactions:

  • UBE2O substrates and interacting partners may differ between tissues

  • Proteomic analysis following UBE2O immunoprecipitation can identify tissue-specific interaction networks

  • Consider examining tissue-specific phenotypes in UBE2O knockout or knockdown models

• Technical considerations for different tissue types:

  • For heart or muscle tissue: Special lysis buffers may be required for efficient protein extraction

  • For brain tissue: UBE2O-BMAL1 interaction has been confirmed in mouse brain lysates, suggesting relevance to neurological studies

  • For liver tissue: UBE2O's role in interferon signaling and potential applications for hepatocellular carcinoma therapy

• Antigen retrieval for immunohistochemistry:

  • For human heart tissue: Use TE buffer pH 9.0 for optimal results

  • Alternative approach: Citrate buffer pH 6.0 may be used for certain tissue types

When studying UBE2O in a new tissue context, preliminary characterization of expression levels and optimization of detection methods are strongly recommended before proceeding with functional studies.

Why might I observe multiple bands or unexpected molecular weights when detecting UBE2O by Western blot?

The detection of multiple bands or unexpected molecular weights for UBE2O is a common technical challenge with several potential explanations:

Understanding these potential sources of variability is crucial for accurate interpretation of UBE2O Western blot results and proper experimental design.

How can I optimize immunohistochemistry protocols for UBE2O detection in tissue samples?

Optimizing immunohistochemistry (IHC) for UBE2O detection requires careful attention to several key parameters:

• Tissue preparation and fixation:

  • Formalin fixation time: Overfixation can mask epitopes; limit to 24-48 hours

  • Sectioning thickness: 4-5 μm sections are generally optimal

  • Mounting: Use positively charged slides to prevent tissue loss during processing

• Antigen retrieval optimization:

  • Primary recommendation: TE buffer pH 9.0

  • Alternative method: Citrate buffer pH 6.0

  • Testing both methods may be necessary for new tissue types

  • Retrieval time and temperature should be optimized (typically 95-100°C for 15-20 minutes)

• Antibody dilution and incubation:

  • Start with the recommended dilution range (1:100-1:400)

  • Perform titration experiments to determine optimal concentration

  • Incubation time and temperature affect sensitivity (overnight at 4°C vs. 1-2 hours at room temperature)

• Detection system selection:

  • Polymer-based detection systems often provide better signal-to-noise ratio

  • DAB substrate development time should be optimized and standardized

  • Consider automated IHC platforms for consistency across multiple samples

• Controls:

  • Human heart tissue has been validated as a positive control for UBE2O IHC

  • Always include negative controls (primary antibody omission) and isotype controls

By systematically optimizing these parameters, researchers can achieve reliable and reproducible UBE2O detection in tissue samples for both research and potential diagnostic applications.