UNC5D Antibody

What is UNC5D and why is it significant in neurobiological research?

UNC5D (Unc-5 Netrin Receptor D) is a transmembrane protein functioning as a netrin receptor that promotes neuronal cell survival and mediates axon guidance. It belongs to the unc-5 family and plays a crucial role in several developmental processes including:

• Axon extension guidance and neuronal growth cone repulsion

• Cell migration during neural development

• Cell-cell adhesion via interaction with FLRT3

• Apoptotic signaling as a dependence receptor when not bound to netrin

UNC5D's involvement in both neural circuit formation and apoptotic regulation makes it a significant target for studying neurodevelopmental disorders and potential implications in cancer metastasis pathology.

What are the molecular characteristics of UNC5D protein that researchers should consider when selecting antibodies?

When selecting UNC5D antibodies, researchers should consider several key molecular characteristics:

• Full-length UNC5D has 953 amino acids with a molecular weight of approximately 106 kDa, though it may appear around 120 kDa on Western blots due to post-translational modifications

• UNC5D can be cleaved by caspases (particularly caspase-2 and caspase-3/7), generating fragments including a C-terminal intracellular domain (UnICD) that can translocate to the nucleus

• Multiple isoforms exist due to alternative splicing

• Post-translational modifications include protein cleavage and glycosylation

• Subcellular localization: primarily cell membrane, though cleaved fragments may localize to the nucleus

These characteristics necessitate careful antibody selection to target appropriate epitopes depending on the research question.

What types of UNC5D antibodies are available and how do they differ in research applications?

Selection considerations include:

• Polyclonal antibodies offer broader epitope recognition but potentially more background

• Antibodies targeting different regions may detect different fragments following caspase cleavage

• C-terminus targeting antibodies are necessary to detect nuclear translocation of cleaved fragments

• Application-specific validation should guide selection for specialized techniques

How should researchers optimize Western blot protocols for detecting UNC5D protein?

Optimizing Western blots for UNC5D requires addressing several technical challenges:

• Sample preparation:

  • For brain/neural tissues: Use ice-cold RIPA buffer with protease inhibitor cocktail

  • Include phosphatase inhibitors if phosphorylation status is relevant

  • Add specific caspase inhibitors (e.g., zVAD-fmk) if preventing cleavage is desired

• Gel selection and running conditions:

  • 8-10% SDS-PAGE gels are optimal for the 106-120 kDa full-length protein

  • 4-20% gradient gels if detecting both full-length protein and cleaved fragments

• Dilution optimization:

  • Start with 1:500-1:2000 dilution range for most UNC5D antibodies

  • Titrate for each specific antibody and tissue type

• Expected band sizes:

  • Full-length: 106-120 kDa

  • Cleaved fragments: Multiple bands including 68 kDa fragment

  • C-terminal fragment: Approximately 40 kDa

• Controls:

  • Positive controls: Mouse brain tissue, mouse kidney tissue, or rat brain tissue

  • Peptide competition assay to confirm specificity

The observed molecular weight may vary (106-120 kDa) due to post-translational modifications, particularly glycosylation patterns that differ between tissue types .

What are the optimal immunostaining protocols for detecting UNC5D in tissue sections and cultured cells?

For successful UNC5D immunostaining:

• Fixation recommendations:

  • Tissue sections: 4% paraformaldehyde fixation for 24 hours

  • Cultured cells: 4% paraformaldehyde for 15 minutes at room temperature

• Antigen retrieval:

  • Heat-mediated retrieval using citrate buffer (pH 6.0) is recommended

  • Pressure cooker: 2-3 minutes at 125°C

• Blocking and permeabilization:

  • Block with 5-10% normal serum from the species of secondary antibody

  • 0.1-0.3% Triton X-100 for permeabilization of cell membranes

  • Include 1% BSA to reduce non-specific binding

• Antibody incubations:

  • Primary antibody dilutions: 1:50-1:200 for IF/IHC applications

  • Incubate overnight at 4°C for optimal results

  • Secondary antibody: 1-2 hours at room temperature

• Special considerations:

  • Double staining with neuronal markers can help contextualize UNC5D expression

  • When studying nuclear translocation, use antibodies recognizing the C-terminus

  • Counterstain nuclei with DAPI to verify nuclear localization of cleaved fragments

These protocols should be optimized based on specific sample types and research questions.

How can UNC5D antibodies be utilized to study protein-protein interactions in the netrin signaling pathway?

To study UNC5D protein interactions:

• Co-immunoprecipitation (Co-IP):

  • Use UNC5D antibodies conjugated to agarose or magnetic beads

  • Verify antibody epitope doesn't interfere with interaction domains

  • Include appropriate controls (IgG, lysate input)

  • Western blot for known interaction partners: DCC, netrins, FLRT3

• Proximity ligation assay (PLA):

  • Requires antibodies from different species for UNC5D and interacting proteins

  • Can detect and quantify transient or weak interactions in situ

  • Particularly useful for studying UNC5D-netrin interactions at cell membranes

• FRET/BRET analysis:

  • Requires tagged proteins but allows live-cell monitoring

  • Useful for studying dynamic regulation of UNC5D interactions

• Crosslinking strategies:

  • Chemical crosslinkers can stabilize transient interactions

  • Follow with immunoprecipitation using UNC5D antibodies

Key interactions to investigate include UNC5D with netrin NTN4, DCC receptor (which may trigger repulsion signaling), and FLRT3 (involved in cell-cell adhesion) .

How can researchers differentiate between full-length UNC5D and its caspase-cleaved fragments using antibodies?

Differentiating UNC5D forms requires strategic antibody selection and analytical approaches:

• Antibody selection strategy:

  • N-terminal-specific antibodies: Detect only full-length protein

  • C-terminal-specific antibodies: Detect both full-length and cleaved C-terminal fragments

  • Middle region antibodies: May detect different fragments depending on cleavage sites

• Subcellular fractionation:

  • Nuclear fractions: Should contain cleaved C-terminal fragments (UnICD)

  • Membrane fractions: Should contain full-length protein

  • Follow with Western blotting using C-terminal antibodies

• Inhibitor experiments:

  • Caspase inhibitors (specific for caspase-2/3/7 or pan-caspase inhibitors like zVAD-fmk) can prevent cleavage

  • Compare protein patterns with/without inhibitors to identify fragments

• Expected fragment patterns:

  • Full-length: ~106-120 kDa

  • Cleaved fragments: Multiple bands including those at position 416, recognized by both caspase-2 and caspase-3/7

  • C-terminal fragment that translocates to the nucleus: ~40 kDa

Researchers should note that UNC5D cleavage occurs at the consensus motif DXXD, with the primary site at position 416 .

What strategies can resolve inconsistent or weak UNC5D antibody detection in neural tissues?

When encountering detection challenges:

• Sample preparation optimization:

  • Fresh tissue yields better results than frozen samples for UNC5D

  • Minimize freeze/thaw cycles which can degrade membrane proteins

  • Use protease inhibitor cocktails specific for neural tissues

• Epitope masking solutions:

  • Try multiple antibodies targeting different regions

  • More stringent antigen retrieval methods for fixed tissues

  • Consider membranous protein extraction buffers containing mild detergents

• Signal enhancement techniques:

  • Tyramide signal amplification for immunohistochemistry

  • Enhanced chemiluminescence substrates for Western blots

  • Increase protein loading (50-100 μg/lane) for low-expression tissues

• Expression level considerations:

  • UNC5D expression is developmentally regulated and may be low in adult tissues

  • Expression is higher in favorable neuroblastomas than unfavorable ones

  • Higher in specific neuronal populations during development

• Appropriate positive controls:

  • Mouse brain tissue, particularly during developmental stages

  • Mouse/rat kidney tissue has been validated as positive control

How can UNC5D antibodies be employed to investigate the role of this receptor in neuronal apoptosis mechanisms?

To study UNC5D's role in apoptosis:

• Co-localization studies:

  • Double immunostaining with UNC5D (C-terminal) antibodies and apoptotic markers

  • Track nuclear translocation of cleaved UNC5D fragments during apoptosis

  • Combine with TUNEL assay to identify apoptotic cells

• Functional analysis in NGF-dependent cells:

  • UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated programmed cell death

  • Monitor UNC5D protein levels during NGF withdrawal using Western blot

  • Combine with functional readouts of apoptosis (caspase activation, PARP cleavage)

• Caspase activation and fragment analysis:

  • Use UNC5D antibodies in conjunction with caspase activity assays

  • Compare wild-type UNC5D and caspase-cleavage site mutants

  • Combined with caspase inhibitors to establish causality

• Netrin-1 dependence studies:

  • UNC5D acts as a dependence receptor required for apoptosis induction when not associated with netrin ligand

  • Compare UNC5D expression and localization in netrin-rich vs. netrin-poor environments

  • Manipulate netrin-1 levels to observe effects on UNC5D-mediated apoptosis

This approach has been particularly informative in neuroblastoma research, where UNC5D expression correlates with favorable outcomes and spontaneous regression .

How should researchers interpret UNC5D expression patterns in relation to neurological disease models?

Interpreting UNC5D expression requires contextual analysis:

• Developmental context:

  • UNC5D expression is dynamically regulated during neural development

  • Compare against normal developmental expression timing in the same tissue

  • Consider co-expression with other netrin receptors (UNC5A-C, DCC)

• Disease-specific considerations:

  • Neuroblastoma: Higher UNC5D expression correlates with favorable outcomes and regression potential

  • UNC5D expression levels significantly segregate patients with neuroblastoma in advanced stages 3 and 4 into those with good and poor prognosis

  • Multivariate analysis indicates UNC5D expression is an independent prognostic factor

• Subcellular localization significance:

  • Membrane localization: Normal receptor function

  • Nuclear localization: Indicates caspase cleavage and potential apoptotic signaling

  • Nuclear UNC5D expression observed in regressing neuroblastoma tissue

• Quantification approaches:

  • Ratio of membrane to nuclear staining

  • Co-localization coefficients with other markers

  • Expression levels relative to appropriate controls

The findings suggest UNC5D may serve as both a biomarker and functional contributor in neurological disease progression or regression.

What are the comparative advantages of different antibody-based techniques for studying UNC5D function in neural development?

Each technique offers distinct advantages for UNC5D research:

Optimal strategy selection depends on specific research questions:

• For developmental studies: IHC/IF in tissue sections with developmental stage comparison

• For mechanistic studies: Combining Western blot for processing with Co-IP for interactions

• For functional studies: Live imaging with targeted mutations or domain deletions

Multiple complementary approaches provide the most comprehensive understanding of UNC5D biology .

How does UNC5D antibody-based research contribute to understanding potential therapeutic targets in neurological disorders and cancer?

UNC5D antibody research has revealed several therapeutic implications:

• Neuroblastoma treatment strategies:

  • UNC5D expression levels significantly correlate with favorable outcomes in neuroblastoma

  • High UNC5D expression is associated with spontaneous regression

  • Potential therapeutic approach: Enhance UNC5D expression or its pro-apoptotic function in aggressive tumors

• Netrin-dependence manipulation:

  • UNC5D acts as a dependence receptor - cells expressing it undergo apoptosis in netrin-poor environments

  • Tumors may downregulate UNC5D to escape dependence-receptor mediated apoptosis

  • Therapeutic strategy: Restore UNC5D expression or block netrin in tumor microenvironments

• Caspase-mediated UNC5D cleavage as therapeutic target:

  • The caspase-released UnICD (UNC5D intracellular domain) translocates to the nucleus and promotes apoptosis

  • Enhancing this process could promote cell death in pathological conditions

  • Conversely, blocking cleavage could promote neuronal survival in neurodegenerative contexts

• NGF-dependency pathway targeting:

  • UNC5D forms a positive feedback loop with p53 and E2F1 to promote NGF dependence-mediated cell death

  • This pathway is crucial for normal neuronal development and tumor suppression

  • Modulating this pathway could address both developmental disorders and cancer

The dual role of UNC5D in both development and apoptosis makes it a particularly interesting target at the intersection of neurology and oncology .

What are the critical methodological considerations when using UNC5D antibodies to study the receptor's role in axon guidance and cell migration?

For studying UNC5D in axon guidance and migration:

• Live versus fixed preparation considerations:

  • Fixed preparations: Capture static snapshots of UNC5D distribution

  • Live imaging: Required for dynamic processes like growth cone guidance

  • Consider photobleaching and phototoxicity effects in live preparations

• In vitro assay optimization:

  • Growth cone turning assays: Require gradient-producing devices

  • Migration assays: Transwell or wound healing with localized netrin sources

  • Require careful controls for mechanical stimulation artifacts

• Spatial gradient considerations:

  • UNC5D function is gradient-dependent (repulsive from netrin sources)

  • Immunostaining should preserve spatial relationships to gradient sources

  • Co-visualization of UNC5D with gradient molecules is essential

• Co-receptor interactions:

  • UNC5D may function independently or with DCC co-receptors

  • Interaction with FLRT3 mediates cell-cell adhesion

  • Antibody epitopes should not interfere with these interaction domains

• Developmental timing:

  • Expression and function are temporally regulated during development

  • Age-matched controls are essential

  • Consider dynamic changes in co-receptor expression

These methodological considerations ensure accurate interpretation of UNC5D's role in the complex processes of axon guidance and cell migration.

How can UNC5D antibodies be utilized in single-cell analysis to understand heterogeneity in neural populations?

Single-cell applications for UNC5D research:

• Single-cell Western blotting:

  • Reveals protein level heterogeneity within neural populations

  • Can detect full-length versus cleaved forms at single-cell resolution

  • Combines with other neuronal markers to identify specific subpopulations

• Mass cytometry (CyTOF):

  • Metal-conjugated UNC5D antibodies for high-parameter analysis

  • Simultaneous detection of UNC5D with dozens of other markers

  • Ideal for complex neural tissue with multiple cell types

• Single-cell immunoprecipitation followed by mass spectrometry:

  • Identifies cell-specific UNC5D interaction partners

  • Reveals potential signaling differences between neural subtypes

  • Can detect post-translational modifications specific to cell states

• Spatial transcriptomics with protein validation:

  • Combine RNA-seq spatial data with UNC5D antibody staining

  • Correlate protein expression with transcriptional programs

  • Preserve spatial context within tissue architecture

These approaches can reveal how UNC5D function varies across neural subtypes and developmental states, potentially explaining differential vulnerability to pathological conditions .

What are the challenges and solutions in developing antibodies that specifically distinguish between UNC5D and its close homologs (UNC5A-C)?

Addressing homolog cross-reactivity challenges:

• Sequence homology considerations:

  • UNC5 family members share significant sequence homology

  • UNC5B is a particularly important paralog of UNC5D

  • Target unique regions for antibody development (low homology areas)

• Validation strategies:

  • Peptide competition assays with specific and homologous peptides

  • Testing on knockout/knockdown models of each family member

  • Western blot comparison with recombinant UNC5A-D proteins

• Domain-specific targeting:

  • Intracellular regions show greater divergence than extracellular domains

  • C-terminal regions often have unique sequences suitable for specific antibody generation

  • Avoid conserved functional domains like death domains

• Cross-reactivity testing panel:

  • Test each antibody against all family members

  • Include positive controls expressing each specific family member

  • Document any cross-reactivity in detailed validation reports

Unlike UNC5D, expression of UNC5A, UNC5B, and UNC5C shows no prognostic significance in primary neuroblastomas, highlighting the importance of specific detection .