UNC5B Antibody

What is UNC5B and what are its alternative names in scientific literature?

UNC5B (Uncoordinated-5 homolog B receptor) is a transmembrane protein initially characterized as a neuronal guidance receptor. In scientific literature, it may be referenced under several alternative names including unc-5 homolog B, UNC5H2, p53RDL1, netrin receptor UNC5B, and p53-regulated receptor for death and life protein 1. Structurally, the UNC5B protein has a molecular mass of approximately 103.6 kilodaltons . When studying UNC5B in the literature, researchers should search using all these alternative names to ensure comprehensive literature review.

Where is UNC5B expressed in mammalian tissues?

UNC5B exhibits widespread expression across multiple tissues beyond its initially characterized role in the nervous system. Research has demonstrated significant UNC5B expression in brain, heart, intestine, blood, lung, kidney, liver, and spleen in murine models . Flow cytometry analysis has specifically identified UNC5B expression on the surface of multiple immune cell populations, including CD15-positive neutrophils, CD45-positive leukocytes, CD14-positive monocytes, and CD3/CD19-positive T and B lymphocytes . When designing experiments targeting UNC5B, researchers should account for this broad expression pattern, particularly when developing tissue-specific interventions.

What detection methods are most effective for UNC5B in different tissue samples?

For UNC5B detection in tissue samples, multiple validated approaches exist depending on research requirements:

• Immunohistochemistry/Immunofluorescence: UNC5B can be detected in fixed paraffin-embedded sections using antigen affinity-purified polyclonal antibodies. Effective protocols involve heat-induced epitope retrieval with basic antigen retrieval reagents, followed by visualization using HRP polymer antibody systems and DAB staining. This approach is particularly effective for localizing UNC5B to specific cellular structures, such as neuronal cell bodies .

• Flow Cytometry: For leukocyte populations, multi-parameter flow cytometry using fluorescently-labeled UNC5B antibodies (typically PE-labeled) in combination with lineage markers (CD45-PerCP, F4/80-APC) provides quantitative analysis of UNC5B expression across different immune cell subsets .

• Western Blotting: This technique provides quantitative analysis of UNC5B protein levels and can validate antibody specificity when analyzing multiple tissue types .

When selecting a detection method, researchers should consider whether they need to assess protein localization, expression levels, or surface versus total cellular expression.

How should researchers validate UNC5B antibody specificity before experimental use?

Rigorous validation of UNC5B antibodies is essential for experimental reliability. A comprehensive validation protocol should include:

• Western Blot Analysis: Confirm the antibody detects a protein band at the expected molecular weight (approximately 103.6 kDa for full-length UNC5B) .

• Immunofluorescence Control Studies: Compare staining patterns between UNC5B-expressing and non-expressing cells/tissues, including appropriate isotype controls (IgG and IgM) to exclude non-specific binding .

• Flow Cytometry Validation: Assess specificity using competitive binding assays and appropriate negative controls to establish specific detection thresholds .

• Functional Validation: For research requiring functional manipulation, confirm that the antibody can modulate UNC5B-dependent processes (such as cell migration) in controlled experimental settings .

Researchers should prioritize antibodies with published validation data across multiple detection platforms to ensure reliable and reproducible results.

What role does UNC5B play in myocardial ischemia-reperfusion injury, and how can antibodies be used to study this pathway?

UNC5B has emerged as a significant contributor to myocardial ischemia-reperfusion (IR) injury through its regulation of leukocyte migration into inflamed cardiac tissue. Studies using UNC5B+/- heterozygous mice demonstrated significantly reduced infarct sizes (23±4% of area at risk) compared to wild-type controls (47±1%), with corresponding reductions in serum troponin I and IL-6 levels .

Researchers can employ UNC5B antibodies to investigate this pathway through several approaches:

• Functional Inhibition Studies: Administration of UNC5B blocking antibodies (125 μg/kg body weight, intravenously) 30 minutes prior to induced ischemia significantly reduced myocardial damage in experimental models .

• Neutrophil Migration Assays: In transendothelial migration assays, UNC5B antibodies significantly inhibit neutrophil migration, providing a mechanistic explanation for the reduced tissue damage in IR injury models .

• Comparative Timing Analysis: The timing of antibody administration appears critical, with different results reported for administration 30 minutes versus 18 hours before ischemia induction, suggesting complex temporal dynamics in UNC5B signaling .

When designing cardiovascular experiments, researchers should consider optimization of antibody dosage (125-800 μg/kg), administration route (intravenous versus intraperitoneal), and timing relative to ischemic events.

How does UNC5B intracellular domain function in cancer biology, and what experimental approaches can assess these mechanisms?

The intracellular domain of UNC5B exhibits paradoxical functions in cancer contexts that challenge the traditional view of UNC5B as primarily pro-apoptotic. In bladder cancer models, the intracellular domain promotes rather than inhibits tumor progression through multiple mechanisms:

• Proliferation Promotion: The intracellular domain of UNC5B promotes cancer cell proliferation in vitro and tumor formation in vivo by interacting with ribosomal proteins .

• Metastatic Enhancement: UNC5B intracellular domain facilitates cell migration, invasion, and metastasis through interactions with fibronectin, beta-catenin, and vimentin .

• Apoptosis Evasion: Despite containing a death domain and caspase-3 cleavage site, the intracellular domain paradoxically fails to activate apoptotic pathways in cancer cells .

Researchers investigating these mechanisms should consider experimental approaches including:

• Truncation studies using UNC5B fragments (residues 399-945, 412-945) to isolate domain-specific functions

• Mass spectrometry to identify protein binding partners of the intracellular domain

• Protein-protein interaction network analysis to identify functional clusters

• Combined in vitro (EDU assay, apoptosis, migration, invasion) and in vivo (xenograft, metastasis) models to comprehensively characterize phenotypic effects

How can UNC5B antibodies be used to study cancer cell migration and metastasis?

UNC5B antibodies provide valuable tools for investigating the complex role of UNC5B in cancer cell motility and metastasis. Experimental approaches include:

• Netrin-1 Pathway Modulation: Monoclonal antibodies targeting the outer-membrane immunoglobulin-like domains of UNC5B can block netrin-1-induced effects on cancer cell mobility. In melanoma A375 cells, such antibodies effectively counteract the inhibitory effect of netrin-1 on cell migration as demonstrated in wound healing and transwell migration assays .

• Pathway Specificity Analysis: Interestingly, while UNC5B antibodies modulate migration, they may not affect cell proliferation (as measured by CCK-8 assay), allowing researchers to dissect distinct UNC5B-mediated pathways .

• Domain-Specific Targeting: For comprehensive understanding, researchers should compare antibodies targeting different UNC5B domains (extracellular vs. intracellular) to distinguish between ligand-dependent and independent functions .

When designing migration studies, researchers should include both 2D (wound healing) and 3D (transwell) assays to fully characterize mobility phenotypes, alongside appropriate controls for antibody specificity.

What are the critical parameters for using UNC5B antibodies in leukocyte migration assays?

When investigating UNC5B's role in leukocyte migration using functional antibodies, several critical parameters require careful optimization:

• Antibody Concentration: Titration experiments should determine optimal concentrations that achieve maximal inhibition without non-specific effects. Published studies have employed concentrations sufficient to significantly reduce transendothelial migration of PMNs .

• Endpoint Assessment: Migration assays should include both direct quantification of migrated cells and assessment of endothelial barrier integrity (using FITC-dextran flux). Importantly, UNC5B antibodies appear to inhibit neutrophil migration without significantly affecting paracellular permeability, suggesting a leukocyte-intrinsic rather than barrier-modulating mechanism .

• Cell-Type Specificity: UNC5B is expressed on multiple leukocyte populations, necessitating cell-type specific assays when investigating migration mechanisms. Flow cytometry confirmation of UNC5B expression on the target cell population should precede migration studies .

• Temporal Dynamics: Time-course experiments are essential as UNC5B-mediated effects may vary depending on acute versus chronic exposure to inhibitory antibodies .

How should researchers interpret conflicting results between genetic and antibody-based UNC5B inhibition approaches?

Researchers may encounter discrepancies between genetic approaches (UNC5B+/- mice, siRNA knockdown) and antibody-based inhibition, requiring careful interpretation:

• Temporal Consideration: Genetic models provide continuous UNC5B inhibition from development, while antibodies offer acute inhibition at specific timepoints. This distinction may explain why UNC5B antibody effects can vary dramatically based on administration timing (30 minutes versus 18 hours pre-insult) .

• Domain Specificity: Antibodies target specific UNC5B domains, potentially inhibiting some but not all UNC5B functions, while genetic approaches affect all domains. In myocardial ischemia models, both UNC5B+/- mice and siRNA knockdown showed concordant protective effects (infarct size reduction from 47±1% to 23±4% and 42±4% to 14±2%, respectively) .

• Compensation Mechanisms: Long-term genetic inhibition may trigger compensatory mechanisms absent in acute antibody inhibition. Performing parallel genetic and antibody studies can help distinguish primary from compensatory effects .

• Dose-Response Relationships: Establish complete dose-response curves for antibodies to determine whether discrepancies with genetic models reflect insufficient dosing rather than true biological differences .

How can UNC5B antibodies help investigate the dual role of UNC5B in apoptosis versus survival pathways?

UNC5B contains structural domains associated with both apoptotic signaling (death domain, caspase-3 cleavage site) and cell survival/proliferation functions, presenting a complex research target:

• Domain-Specific Antibodies: Researchers can generate antibodies targeting specific UNC5B domains (death domain, caspase-3 cleavage site at position 412) to dissect their individual contributions to cell fate decisions .

• Context-Dependent Function: Studies in bladder cancer have revealed that the intracellular domain fails to activate apoptosis despite containing canonical death domains, suggesting context-dependent regulation. Researchers should examine UNC5B function across multiple cell types using the same antibody to identify context-specific modulators .

• Protein Interaction Mapping: Mass spectrometry combined with protein-protein interaction networks can reveal how UNC5B binding partners differ between apoptotic and proliferative contexts. Antibodies can confirm these interactions through co-immunoprecipitation approaches .

• Caspase Activation Analysis: Despite containing a caspase-3 cleavage site, the UNC5B intracellular domain does not activate caspase-3 in bladder cancer cells. Researchers should investigate this paradox using antibodies that specifically recognize the cleaved form versus intact UNC5B .

The complex, context-dependent functions of UNC5B highlight the importance of comprehensive experimental design that integrates multiple antibody-based approaches with genetic and pharmacological validation strategies.