RUNDC3B Antibody

What is RUNDC3B and why is it relevant to cancer research?

RUNDC3B (RUN Domain Containing 3B) is a protein that contains a RUN domain in its N-terminal region, which mediates interaction with Rap2, an important component of the mitogen-activated protein kinase (MAPK) cascade. This cascade regulates cellular proliferation and differentiation processes critical to cancer development. RUNDC3B is expressed in multiple tissues including brain, thymus, ovary, testis, leukocytes, liver, small intestines, and prostate . Its relevance to cancer research stems from its differential methylation patterns observed between lymphoid and myeloid malignancies, with potential applications as a biomarker, particularly in lymphoid malignancies .

How does RUNDC3B function in normal cellular processes?

RUNDC3B is predicted to interact with Rap2, similar to its homolog RUNDC3A. The Rap protein family constitutes a subgroup of the Ras superfamily of small GTPases that function as molecular switches regulating cellular functions including proliferation, differentiation, and cell motility . RUNDC3B likely serves as a mediator between Rap2 and the MAPK signaling cascade, as its protein sequence contains characteristic binding sites for MAPK intermediates . This connection to fundamental cellular pathways explains why dysregulation of RUNDC3B may contribute to pathological processes.

What is the relationship between RUNDC3B expression and DNA methylation?

DNA methylation in the RUNDC3B promoter region is inversely associated with gene expression. Studies have demonstrated that increased methylation density in the CpG island of the RUNDC3B promoter correlates significantly with decreased expression (ρ = 0.77, p < 0.001) . This epigenetic silencing mechanism appears particularly relevant in lymphoid malignancies, where treatment with demethylating agents has been shown to restore RUNDC3B expression . Researchers working with RUNDC3B antibodies should consider the methylation status of their experimental models when interpreting expression data.

What are the primary applications for RUNDC3B antibodies in cancer research?

RUNDC3B antibodies are primarily used to:

• Detect protein expression patterns across different cancer types and normal tissues

• Investigate relationships between epigenetic modifications and protein expression

• Study alterations in RUNDC3B expression in drug-resistant cancer cells

• Evaluate potential biomarker applications in lymphoid versus myeloid malignancies

• Explore connections between RUNDC3B and MAPK pathway dysregulation in oncogenesis

How does RUNDC3B expression differ between drug-resistant and drug-sensitive cancer cell lines?

RUNDC3B shows significant upregulation in multiple drug-resistant cancer cell lines. According to microarray expression data, RUNDC3B is overexpressed in paclitaxel-resistant cell lines (A2780PR1, A2780PR2, and W1PR) with fold changes of 13.07, 56.57, and 10.26 respectively . It is also upregulated in doxorubicin-resistant cell lines, particularly A2780DR1 (8.56-fold) and W1DR (17.55-fold) . This pattern suggests RUNDC3B may play a role in mechanisms of drug resistance, potentially through its interaction with signaling pathways that regulate cell survival and apoptosis. When designing experiments with RUNDC3B antibodies in drug resistance studies, researchers should consider these differential expression patterns and include appropriate controls.

What are the challenges in distinguishing RUNDC3B from its homolog RUNDC3A when using antibodies?

One significant challenge in RUNDC3B antibody research is ensuring specificity against its homolog RUNDC3A. Both proteins share high homology and interact with Rap2 , which may lead to cross-reactivity issues with antibodies. Researchers should:

• Thoroughly validate antibody specificity using positive and negative controls

• Consider using knockout or knockdown models as antibody validation controls

• Employ multiple detection methods (Western blot, immunohistochemistry, immunofluorescence) with different antibodies targeting distinct epitopes

• Verify results with transcript-level analysis (RT-PCR) to distinguish between the homologs

• Check antibody datasheets for cross-reactivity testing against RUNDC3A

What is the significance of region-specific methylation in the RUNDC3B promoter for protein expression?

Specific regions within the RUNDC3B CpG island demonstrate stronger associations with gene silencing than others. Statistical analysis using odds ratios showed that regions 2, 3, 4, 5, and 6 of the promoter CpG island exhibited significant inverse associations between methylation and expression . The strongest associations were observed in regions 3 (OR: 135, 95% CI: 4.87–3744.64, p = 0.004) and region 6 (OR: 141.67, 95% CI: 5.14–3907.44, p = 0.004) . When investigating RUNDC3B expression using antibodies, researchers should consider correlating protein levels with region-specific methylation data, particularly focusing on these highly significant regions, to better understand epigenetic regulation mechanisms.

How does RUNDC3B relate to transcriptional fusion events in drug-resistant cancers?

While the search results don't directly link RUNDC3B to fusion events, they mention that drug-resistant cancer cells frequently exhibit transcriptional fusions involving ABCB1, a major drug resistance gene that is co-expressed with RUNDC3B in certain resistant cell lines . This suggests potential regulatory relationships or coordinated expression patterns between these genes in drug resistance mechanisms. Researchers should consider investigating whether RUNDC3B participates in or is affected by fusion events or chromosome rearrangements in drug-resistant cells, which may require specialized antibody applications beyond standard expression analysis.

What are the optimal conditions for Western blot detection of RUNDC3B?

Based on comparable protein detection methodologies described in the literature, the following protocol is recommended for RUNDC3B Western blotting:

• Prepare whole-cell lysates using RIPA buffer containing protease inhibitor cocktail

• Sonicate samples to ensure complete lysis and protein extraction

• Determine protein concentration using DC™ protein assay or equivalent

• Load 40-50 μg of protein lysate on a 4-20% gradient polyacrylamide gel

• Perform gel electrophoresis at 150V for approximately 1 hour

• Transfer proteins using a high molecular weight transfer protocol (similar to that used for MDR1/ABCB1)

• Block membranes in Odyssey Blocking Buffer or 5% non-fat milk in TBS-T for 1 hour

• Incubate with primary RUNDC3B antibody (1:1000 dilution) overnight at 4°C

• Wash three times with TBS-T

• Incubate with appropriate secondary antibody (1:15,000 dilution) for 1 hour at room temperature

• Wash three times with TBS-T before imaging

Since RUNDC3B levels may be low in some tissues or cell lines, researchers should consider extended exposure times and highly sensitive detection methods.

How can researchers validate the specificity of RUNDC3B antibodies?

To ensure antibody specificity for RUNDC3B:

• Knockout/knockdown validation: Test antibody in RUNDC3B knockout or siRNA-mediated knockdown samples

• Peptide competition: Pre-incubate antibody with the immunizing peptide to block specific binding

• Overexpression validation: Test in cells overexpressing tagged RUNDC3B and confirm co-localization

• Multiple antibody comparison: Use antibodies targeting different epitopes of RUNDC3B

• Cross-reactivity assessment: Test against recombinant RUNDC3A to ensure no cross-reactivity with the homolog

• Tissue specificity: Confirm expression patterns match known tissue distribution of RUNDC3B

What controls should be included when measuring RUNDC3B expression in methylation studies?

When investigating relationships between RUNDC3B methylation and expression:

• Cell line controls: Include cell lines with known RUNDC3B methylation status (e.g., lymphoid vs. myeloid cell lines)

• Treatment controls: Use samples treated with demethylating agents (e.g., 5-aza-2'-deoxycytidine) as positive controls for expression restoration

• Normal tissue controls: Include normal cell counterparts to cancer cells being studied

• Housekeeping controls: Normalize protein expression using stable housekeeping proteins (GAPDH, β-actin)

• Transcript level controls: Perform parallel RT-PCR for RUNDC3B transcript to correlate with protein levels

• Region-specific methylation controls: Include samples with varying methylation patterns across different promoter regions

What methodological approaches can address contradictory RUNDC3B expression data?

When faced with inconsistent RUNDC3B expression results:

• Multiple detection methods: Combine Western blot, immunohistochemistry, and RT-PCR

• Antibody validation: Re-validate antibody specificity using the methods described in FAQ 3.2

• Quantification method assessment: Compare different protein quantification approaches

• Subcellular fractionation: Determine if RUNDC3B localization varies between samples

• Post-translational modification analysis: Investigate whether modifications affect antibody recognition

• Statistical approaches: Apply appropriate statistical methods similar to those used in RUNDC3B methylation studies (e.g., Spearman's rank correlation, odds ratios)

• Methylation correlation: Assess whether contradictory protein expression data correlates with methylation patterns

How can RUNDC3B antibodies be used to investigate lymphoid malignancies?

RUNDC3B antibodies offer valuable applications in lymphoid malignancy research:

• Differential diagnosis: Distinguish lymphoid from myeloid malignancies based on RUNDC3B expression patterns

• Biomarker validation: Evaluate RUNDC3B protein levels in correlation with methylation status as a potential biomarker

• Treatment response monitoring: Assess RUNDC3B expression changes following demethylating therapy

• Prognostic indicator development: Investigate correlations between RUNDC3B levels and clinical outcomes

• Pathway analysis: Study RUNDC3B's interactions with MAPK pathway components in lymphoid cancer progression

• Therapy resistance: Evaluate RUNDC3B expression in relation to treatment resistance, as suggested by its upregulation in drug-resistant cell lines

What is the significance of RUNDC3B upregulation in drug-resistant cancer cell lines?

The consistent upregulation of RUNDC3B across multiple drug-resistant cell lines (see Table 1) suggests its potential involvement in drug resistance mechanisms .

Table 1: RUNDC3B Expression Changes in Drug-Resistant Cancer Cell Lines

Data adapted from Table 1 in search result

Researchers using RUNDC3B antibodies in drug resistance studies should:

• Investigate correlations between RUNDC3B and known drug resistance proteins (e.g., ABCB1, ABCG2)

• Examine whether RUNDC3B knockdown affects drug sensitivity

• Study subcellular localization changes of RUNDC3B in resistant versus sensitive cells

• Explore whether RUNDC3B interacts with drug efflux pumps or drug metabolism enzymes

• Consider RUNDC3B as a potential therapeutic target to overcome resistance

How can RUNDC3B antibodies be used to study its interaction with the MAPK pathway?

To investigate RUNDC3B's role in MAPK signaling:

• Co-immunoprecipitation: Use RUNDC3B antibodies to pull down protein complexes and identify MAPK pathway components

• Proximity ligation assays: Visualize and quantify interactions between RUNDC3B and Rap2 or other MAPK components

• Immunofluorescence co-localization: Determine subcellular regions where RUNDC3B and MAPK components interact

• Pathway activation analysis: Correlate RUNDC3B expression with phosphorylation status of MAPK pathway members

• Knockout/knockdown studies: Assess MAPK pathway activity in cells with altered RUNDC3B expression

• Domain-specific antibodies: Use antibodies targeting specific domains of RUNDC3B to understand which regions are critical for MAPK interactions

Given that RUNDC3B is predicted to interact with Rap2 and contains binding sites for MAPK intermediates , these approaches could help elucidate its precise role in signaling.

What are the challenges in correlating RUNDC3B protein levels with methylation status?

Researchers face several challenges when correlating RUNDC3B protein levels with methylation:

• Region-specific effects: Different promoter regions show varying strength of association with expression

• Partial methylation complexities: Interpreting the effects of partial methylation versus complete methylation

• Cell type variations: Different cell types may exhibit different methylation-expression relationships

• Temporal dynamics: Changes in methylation may not immediately reflect in protein levels

• Post-transcriptional regulation: Other regulatory mechanisms may override methylation effects

• Antibody sensitivity limitations: Low expression levels might be difficult to detect precisely

• Technical variability: Different methylation detection methods may yield varying results

Researchers should consider these factors when designing experiments that correlate RUNDC3B methylation with protein expression.

How should researchers interpret discrepancies between RUNDC3B transcript and protein levels?

When faced with disconnects between RUNDC3B mRNA and protein levels:

• Post-transcriptional regulation: Investigate microRNA-mediated regulation or RNA stability factors

• Protein stability assessment: Examine protein half-life using protein synthesis inhibitors

• Proteasomal degradation: Test if proteasome inhibitors affect RUNDC3B protein levels

• Alternative splicing: Check for splice variants that might not be detected by standard antibodies

• Technical considerations: Verify the linearity and sensitivity ranges of both RT-PCR and antibody-based methods

• Statistical approach: Apply correlation analysis similar to methylation density studies to quantify transcript-protein relationships

• Time-course experiments: Account for temporal delays between transcription and translation

What experimental designs are optimal for investigating RUNDC3B in drug resistance mechanisms?

For researching RUNDC3B's role in drug resistance:

• Paired sensitive/resistant models: Use matched cell line pairs that differ in drug resistance status

• Gradient resistance models: Develop models with increasing resistance levels to correlate with RUNDC3B expression

• Knockdown/overexpression studies: Manipulate RUNDC3B levels and assess impact on drug sensitivity

• In vitro/in vivo correlation: Validate cell line findings in patient-derived xenografts or clinical samples

• Multi-drug resistance assessment: Test whether RUNDC3B expression correlates with resistance to multiple drug classes

• Combination with other markers: Study RUNDC3B alongside established resistance markers like ABCB1

• Reversal strategies: Investigate whether targeting RUNDC3B (or its methylation) can restore drug sensitivity

What statistical approaches are recommended for analyzing RUNDC3B antibody data in methylation studies?

Based on established approaches in RUNDC3B methylation research , recommended statistical methods include:

• Methylation density scoring: Calculate weighted averages across different methylation regions

• Spearman's rank correlation: Assess relationships between ranked methylation scores and expression levels

• Odds ratio calculations: Determine strength of associations between methylation status and expression levels

• Confidence interval reporting: Include 95% confidence intervals with odds ratios

• Categorical data analysis: Use appropriate statistics for unmethylated/partially methylated/methylated classifications

• Multiple region analysis: Analyze each promoter region separately to identify critical regulatory areas

• Multivariate analysis: Control for confounding factors when analyzing clinical samples

How can researchers optimize RUNDC3B antibody-based assays for clinical biomarker applications?

To develop RUNDC3B as a clinical biomarker:

• Antibody standardization: Validate specific antibody clones across multiple laboratories

• Tissue processing optimization: Develop standardized protocols for tissue preparation

• Quantification standardization: Establish scoring systems for immunohistochemistry

• Reference range determination: Define normal expression ranges across relevant tissues

• Clinical correlation studies: Associate RUNDC3B levels with patient outcomes

• Combination biomarker panels: Evaluate RUNDC3B alongside other markers for improved predictive value

• Comparative methodology studies: Assess agreement between antibody-based and methylation-based detection

• Reproducibility assessment: Conduct inter-laboratory and inter-observer variability studies

What are emerging applications for RUNDC3B antibodies in precision medicine?

RUNDC3B antibodies show promise for several precision medicine applications:

• Diagnostic stratification: Distinguishing lymphoid from myeloid malignancies

• Treatment selection: Identifying patients who might benefit from demethylating agents

• Resistance prediction: Screening for potential drug resistance based on RUNDC3B overexpression patterns

• Therapeutic target validation: Developing targeted approaches against RUNDC3B or its regulated pathways

• Minimal residual disease monitoring: Tracking RUNDC3B expression in treated patients

• Clinical trial stratification: Using RUNDC3B status to select patients for specific therapeutic approaches

• Combination therapy rationale: Developing strategies to target RUNDC3B alongside conventional treatments

What are priority research questions regarding RUNDC3B that remain unanswered?

Critical knowledge gaps that require further investigation include:

• Mechanistic role in drug resistance: How exactly does RUNDC3B contribute to resistance phenotypes?

• Functional consequences of methylation: Beyond correlation, what are the biological effects of RUNDC3B methylation?

• Protein interactions: What is the complete interactome of RUNDC3B beyond predicted Rap2 binding?

• Tissue-specific functions: How does RUNDC3B function differ across its various expression sites?

• Post-translational modifications: What modifications affect RUNDC3B function and detection?

• Clinical utility validation: Does RUNDC3B expression or methylation have demonstrable clinical value?

• Therapeutic targeting potential: Can RUNDC3B itself be effectively targeted for therapy?