UNC5A Antibody

What criteria should researchers consider when selecting an UNC5A antibody for their experiments?

When selecting an UNC5A antibody, researchers should consider:

• Target specificity: Verify the antibody specifically recognizes UNC5A (also known as UNC5H1) without cross-reactivity to other UNC5 family members (UNC5B, UNC5C, UNC5D) .

• Species reactivity: Confirm compatibility with your experimental model. Many UNC5A antibodies react with human, mouse, and rat samples, while some offer broader reactivity .

• Applications: Ensure the antibody is validated for your specific application (Western blot, immunofluorescence, flow cytometry, ELISA, etc.) .

• Clonality: Choose between polyclonal antibodies (offering multiple epitope recognition) or monoclonal antibodies (providing greater specificity and reproducibility) .

• Epitope region: Select antibodies targeting specific regions of interest (extracellular domain, death domain, etc.) based on your research questions .

• Conjugation: Consider whether you need unconjugated antibodies or those conjugated to specific tags (FITC, biotin, etc.) .

How should researchers validate UNC5A antibody specificity before experimental use?

Thorough validation should include:

• Positive and negative controls: Use cell lines or tissues known to express or lack UNC5A expression .

• Knockdown/knockout verification: Compare antibody signal in UNC5A knockdown/knockout samples versus controls to confirm specificity .

• Multiple detection methods: Validate across different techniques (e.g., Western blot, immunofluorescence, flow cytometry) .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to confirm specificity through signal reduction .

• Molecular weight verification: Confirm detection at the expected molecular weight (approximately 93 kDa for UNC5A) .

Proper validation is particularly crucial as UNC5A has multiple isoforms and shares homology with other UNC5 family members, which could lead to cross-reactivity issues.

How can UNC5A antibodies be used to study neurite outgrowth processes?

UNC5A antibodies enable various neurodevelopmental research approaches:

• Localization studies: Immunofluorescence using UNC5A antibodies can reveal subcellular localization during neurite outgrowth, particularly at regions of neuronal differentiation .

• Co-localization experiments: Combined with markers for FANCC or other proteins involved in neurite outgrowth, UNC5A antibodies help determine spatial relationships during neuronal development .

• Differentiation markers: UNC5A antibodies can track expression changes during neuronal differentiation processes, particularly after retinoic acid or Netrin-1 treatment .

• Temporal expression patterns: Western blot analysis using UNC5A antibodies can quantify expression levels at different developmental stages .

Researchers have demonstrated that UNC5A co-localizes with FANCC protein at regions of neurite outgrowth during neuronal cell differentiation, suggesting an important role in axonal-like growth processes .

What experimental approaches can determine the interaction between UNC5A and Netrin-1 in axonal pathfinding?

Several methodological approaches using UNC5A antibodies can elucidate this interaction:

• Co-immunoprecipitation: Use UNC5A antibodies to pull down protein complexes and analyze for Netrin-1 presence .

• Proximity ligation assays: Detect in situ protein-protein interactions between UNC5A and Netrin-1 at subcellular resolution.

• Structural binding studies: Surface plasmon resonance (SPR) combined with immunological detection can measure binding kinetics between UNC5A and Netrin-1 .

• Heparin binding assays: SPR measurements have shown that UNC5A binds heparin via a positively charged patch at the boundary of its IG1 and IG2 domains, with mutations at this site affecting both heparin and UNC-6/netrin binding .

Recent structural studies have provided insights into the formation of repulsive netrin guidance signaling complexes, showing that UNC5A contains two immunoglobulin domains (IG1 and IG2) with flexibility at their boundary, which may be necessary for recognition by heparan sulfate and protein ligands .

How does UNC5A expression correlate with clinical outcomes in breast cancer, and what experimental approaches can verify this relationship?

UNC5A appears to have important prognostic significance in breast cancer:

Detailed studies show that knockdown of UNC5A in ER+ breast cancer cells leads to altered basal gene expression affecting plasma membrane integrity and ERα signaling, resulting in ligand-independent activity of ERα, altered turnover of phosphorylated ERα, and E2-independent tumorigenesis with multiorgan metastases .

What molecular mechanisms link UNC5A downregulation to cancer progression, and how can researchers study these pathways?

Multiple mechanisms have been identified linking UNC5A to cancer progression:

• EGFR signaling pathway: UNC5A knockdown increases EGFR protein levels (but not mRNA) and enhances AKT activation. This relationship can be studied through:

  • Western blot analysis of EGFR levels in UNC5A-depleted cells

  • Phosphorylation studies of downstream effectors like AKT and ERK

  • Correlation analysis between UNC5A and EGFR expression in tumor samples

• Epigenetic regulation: UNC5A expression can be epigenetically downregulated through O-GlcNAcylation-dependent mechanisms involving EZH2:

  • Cut&Run experiments to analyze EZH2 binding to the UNC5A promoter

  • Luciferase promoter activity assays to measure UNC5A transcriptional regulation

  • siRNA knockdown of OGT (O-GlcNAc transferase) to assess effects on UNC5A expression

• Cell phenotype transition: UNC5A depletion leads to a luminal/basal hybrid phenotype in breast cancer:

  • Analysis of markers including ΔNp63, CD44, CD49f, EGFR, and NTN4

  • Assessment of luminal/alveolar differentiation-associated ELF5 expression

  • Evaluation of bipotent luminal progenitor characteristics (KRT14+/KRT19+ and CD49f+/EpCAM+)

How can researchers optimize UNC5A antibody staining for dual immunofluorescence protocols in neural tissues?

Optimizing dual immunofluorescence with UNC5A antibodies requires careful protocol adjustments:

• Fixation optimization:

  • For cultured neurons: 4% paraformaldehyde for 15-20 minutes at room temperature

  • For neural tissues: 4% paraformaldehyde with subtle permeabilization using 0.1-0.3% Triton X-100

• Antibody selection:

  • Choose UNC5A antibodies raised in species different from your second target antibody

  • For difficult co-staining, consider directly conjugated UNC5A antibodies (FITC, AbBy Fluor® 488/555)

• Signal amplification strategies:

  • For weak UNC5A signals, employ tyramide signal amplification

  • Sequential staining may reduce cross-reactivity issues

• Controls to include:

  • Single antibody controls to assess bleed-through

  • UNC5A knockdown samples to verify specificity

  • Secondary antibody-only controls to evaluate background

  • Absorption controls using blocking peptides

When studying UNC5A co-localization with FANCC in SH-SY5Y cells, researchers successfully used retinoic acid (10 μM) treatment for 48 hours or recombinant human Netrin-1 (500 ng/ml) treatment for 4 hours prior to immunofluorescence staining .

What approaches can resolve contradictory Western blot data when detecting UNC5A in different cell types?

When facing contradictory UNC5A Western blot results across different cell types, consider these systematic troubleshooting approaches:

• Protein extraction optimization:

  • For transmembrane proteins like UNC5A, compare different lysis buffers (RIPA vs. NP-40)

  • Include appropriate protease inhibitors to prevent degradation

  • Consider membrane protein enrichment protocols

• Expression level variations:

  • UNC5A expression is highly context-dependent and can vary significantly between tissues

  • Expression can be regulated by p53 status, exposure to chemotherapeutic drugs, and O-GlcNAcylation

  • Verify expression patterns using RT-qPCR in parallel with protein detection

• Antibody epitope accessibility:

  • Test multiple UNC5A antibodies targeting different domains

  • Use recommended dilution ranges (typically 1:500-1:1000 for Western blot)

  • Consider native vs. denaturing conditions affecting epitope exposure

• Post-translational modifications:

  • UNC5A undergoes O-GlcNAcylation and potentially other modifications

  • These modifications may affect antibody binding in tissue-specific contexts

  • Use phosphatase or glycosidase treatments to investigate these effects

How can researchers investigate the structural basis of UNC5A interactions with netrin guidance complexes?

Advanced structural studies of UNC5A interactions can employ multiple complementary approaches:

• Crystallographic analysis:

  • Current structural data shows UNC5A contains two immunoglobulin domains (IG1 and IG2)

  • The IG2 domain is N-glycosylated at a conserved site (Asn178 on the F strand)

  • The angle between IG1 and IG2 domains shows flexibility, which may be necessary for ligand recognition

• Yeast surface display systems:

  • Established platforms can measure UNC-6/netrin-UNC-5 interactions

  • Directed evolution experiments have identified key residues (N18 in IG1 and N188 in IG2) involved in binding

• Surface plasmon resonance (SPR):

  • SPR measurements have shown that UNC-5 binds heparin with a Kd of 26 μM

  • Mutations N18K and N188K improve heparin binding 16-fold (Kd of 1.7 μM)

  • UNC-6ΔC binds heparin with much higher affinity (Kd of 0.04 μM)

• Mutagenesis studies:

  • Mutations at the heparin-binding site can strengthen or weaken UNC-5–UNC-6 interactions

  • The N18K and N188K mutations improve binding to UNC-6

What methodological approaches can determine whether UNC5A functions as a dependence receptor in specific cellular contexts?

To establish UNC5A's function as a dependence receptor in different contexts:

• Cell survival assays:

  • Compare cell viability in UNC5A-expressing and UNC5A-knockdown cells under varying Netrin-1 concentrations

  • Assess apoptosis markers (caspase activation, PARP cleavage) in response to Netrin-1 withdrawal

  • Evaluate how FANCC interaction with UNC5A delays UNC5A-mediated apoptosis

• Domain-specific functional analysis:

  • Use UNC5A constructs with mutations in the death domain to assess apoptotic function

  • Examine how FANCC interaction with UNC5A's cytoplasmic death domain affects apoptotic signaling

• Ligand-receptor dynamics:

  • Evaluate UNC5A-mediated apoptosis independently of Netrin-1, suggesting other functional ligands

  • Investigate how p53-dependent upregulation of UNC5 receptors and Netrin-1 affects cell survival

• Therapeutic applications:

  • Explore how Netrin-1/UNC5A interaction inhibitors (like TRAP-netrin UNC5A) might potentiate chemotherapy-induced cell death

  • Investigate whether targeting OGT/EZH2 interaction enhances chemotherapy efficiency in colorectal cancer by affecting UNC5A expression