SLITRK5 Antibody

What is SLITRK5 and what are its key structural features?

SLITRK5 (SLIT and NTRK-like family member 5) is a transmembrane protein with a canonical length of 958 amino acid residues and a molecular mass of 107.5 kDa in humans. It belongs to the SLITRK protein family consisting of six homologous transmembrane proteins (SLITRK1-6) .

The protein structure includes:

• Two conserved leucine-rich repeat (LRR) domains in the extracellular region

• Each LRD (aa 80-285 and aa 384-579) contains seven and eight leucine-rich repeats, respectively

• A single-pass type I membrane domain

• An intracellular region with two conserved potential phosphorylation sites (Tyr833 and Tyr917)

• A TrkA-like tyrosine phosphorylation site (YLxxL) at Tyr945

SLITRK5 shares structural homology with Slit (in its extracellular domain) and with Trk neurotrophin receptors (in its intracellular domain) .

What is the expression pattern and localization of SLITRK5?

SLITRK5 exhibits tissue-specific expression with the following characteristics:

• Highly expressed in the cerebral cortex of the brain

• Lower expression levels in the spinal cord and medulla

• Present in the pyramidal layer of the hippocampus and thalamus during embryonic development

• Subcellular localization is in the cell membrane

This restricted expression pattern suggests specialized functions in neural tissues, distinguishing it from the broader expression profiles of some other membrane proteins.

What are the primary applications for SLITRK5 antibodies in research?

SLITRK5 antibodies are employed in multiple experimental techniques:

These applications allow researchers to detect, localize, and quantify SLITRK5 expression in different experimental contexts .

How should I optimize Western blot protocols for SLITRK5 detection?

For optimal Western blot results with SLITRK5 antibodies:

• Sample preparation:

  • Include protease inhibitors in lysis buffers to prevent degradation

  • Use appropriate detergents for membrane protein extraction (e.g., RIPA buffer)

• Gel selection and protein loading:

  • Use 8-10% SDS-PAGE gels due to SLITRK5's high molecular weight (107 kDa)

  • Expected band size may vary (100-160 kDa) due to post-translational modifications like glycosylation

• Transfer and blocking:

  • Employ longer transfer times for large proteins

  • Use 5% BSA in TBS-T for blocking to reduce background

• Antibody incubation:

  • Start with a 1:1000 dilution and optimize as needed

  • Consider overnight primary antibody incubation at 4°C

• Positive controls:

  • HEK-293 or NIH-3T3 cell lysates are recommended as positive controls

What factors should I consider when selecting SLITRK5 antibodies?

When choosing a SLITRK5 antibody, evaluate these critical parameters:

• Antibody type:

  • Polyclonal antibodies provide broader epitope recognition

  • Monoclonal antibodies offer higher specificity for particular domains

• Host species:

  • Consider compatibility with your experimental system

  • Rabbit-derived antibodies are commonly available for SLITRK5

• Target epitope:

  • N-terminal antibodies target the extracellular domain

  • C-terminal antibodies recognize the intracellular region

  • Some antibodies are specifically raised against the LRR domains

• Reactivity:

  • Verify cross-reactivity with your species of interest

  • Most SLITRK5 antibodies react with human and mouse samples

  • Some also recognize rat, dog, cow, sheep, pig, and horse SLITRK5

• Validation data:

  • Review Western blot images provided by manufacturers

  • Check citation records for successful applications in published research

How does SLITRK5 function in neuronal development?

SLITRK5 plays crucial roles in neuronal development through several mechanisms:

• Neurite outgrowth regulation:

  • Unlike SLITRK1, SLITRK5 inhibits neurite outgrowth in cultured neurons

  • This suggests a role in constraining or directing neuronal process extension

• Synaptic connectivity:

  • Functions in chemical synaptic transmission

  • Mediates trans-synaptic interactions with presynaptic receptor-type protein tyrosine phosphatases δ (PTPδ)

  • SLITRK5 binds PTPδ through its LRR1 domain

• Corticostriatal circuitry:

  • SLITRK5 deficiency impairs corticostriatal connectivity

  • SLITRK5 knockout mice display obsessive-compulsive–like behaviors

  • Deficiency leads to decreased protein amounts of glutamate receptor subunits NR2A, NR2B, GluR1, and GluR2

These findings position SLITRK5 as a critical regulator of neural circuit formation and function, with implications for neuropsychiatric disorders.

What is SLITRK5's role in hedgehog signaling and bone formation?

SLITRK5 functions as a negative regulator of hedgehog (Hh) signaling in osteoblasts:

• Osteoblast differentiation:

  • SLITRK5-deficient osteoblasts show enhanced differentiation and increased mineralization

  • Knockout cells display increased alkaline phosphatase (ALP) activity

  • Expression of osteoblast marker genes (Runx2, Sp7, Bsp, Ocn, Alpl) is markedly increased in Slitrk5−/− osteoblasts

• Molecular mechanism:

  • SLITRK5 binds directly to Sonic Hedgehog (SHH) through its extracellular domain

  • It also interacts with PTCH1 through its intracellular domain

  • Surface plasmon resonance shows binding of SLITRK5 extracellular domain to SHH with a Kd of ~40 nM

  • This interaction is specific to SLITRK5 and not observed with other SLITRK family members (SLITRK1, SLITRK6)

• Therapeutic potential:

  • SLITRK5 may represent an attractive therapeutic target for enhancing bone formation

  • Inhibition of SLITRK5 could potentially promote osteoblast differentiation and bone formation

How does SLITRK5 interact with neurotrophin signaling pathways?

SLITRK5 mediates BDNF-dependent TrkB receptor trafficking through specific interactions:

• TrkB receptor binding:

  • SLITRK5 interacts specifically with TrkB receptor

  • This interaction is not observed with other Slitrk members (Slitrk1-3)

  • It is also not seen with TrkC, another major CNS neurotrophin receptor

• BDNF-dependent modulation:

  • Serum starvation significantly reduces the basal interaction between SLITRK5 and TrkB

  • BDNF stimulation significantly increases the interaction between SLITRK5 and TrkB

  • This enhancement is blocked by pretreatment with K252a, an inhibitor of Trk kinases

• Domain mapping:

  • The interaction between SLITRK5 and TrkB is mediated by specific domains

  • Chimeric constructs swapping domains between SLITRK5 and SLITRK1 have been used to map the interaction domains

This suggests SLITRK5 functions in neurotrophin signaling regulation, potentially influencing neuronal survival, differentiation, and plasticity.

What experimental approaches can be used to study SLITRK5-protein interactions?

Several methodologies have proven effective for investigating SLITRK5 interactions:

• Co-immunoprecipitation (Co-IP):

  • Demonstrates interactions between SLITRK5 and binding partners like SHH, PTCH1, and TrkB

  • Can be performed bidirectionally using tagged constructs (e.g., Flag-SLITRK5 and HA-PTCH1)

  • Allows assessment of domain-specific interactions using truncation mutants

• Cell-free binding assays:

  • Recombinant SLITRK5 extracellular domain can be affixed to solid phase

  • Interaction with epitope-tagged proteins (e.g., His-SHH) can be detected via anti-His HRP

  • Enables direct interaction studies outside cellular context

• Surface plasmon resonance:

  • Provides quantitative binding kinetics data

  • Has been used to determine binding affinity between SLITRK5 and SHH (Kd ~40 nM)

• Binding assays with soluble protein domains:

  • Exposure of cells expressing SLITRK5 to soluble purified protein domains (e.g., PTPδ-Fc)

  • Useful for studying trans-interactions with cell surface receptors

• Chimeric protein approaches:

  • Swapping domains between SLITRK family members

  • Helps map specific interaction domains within the protein

How can I optimize immunofluorescence detection of SLITRK5 in neural tissues?

For optimal immunofluorescence visualization of SLITRK5 in neural tissues:

• Tissue preparation:

  • Fresh frozen sections preserve epitope accessibility

  • For fixed samples, use 4% PFA with short fixation times (10-15 minutes)

  • Consider antigen retrieval methods if necessary

• Antibody selection and dilution:

  • Start with 1:200-1:800 dilution range for primary antibodies

  • Use antibodies validated for immunofluorescence applications

  • Consider antibodies targeting different epitopes of SLITRK5

• Signal amplification:

  • TSA (tyramide signal amplification) can enhance detection of low-abundance proteins

  • Secondary antibody selection should match experimental design

• Controls:

  • Include SLITRK5-deficient tissues as negative controls

  • Use cerebral cortex sections as positive controls due to high expression

  • Consider co-staining with neuronal markers for colocalization studies

• Imaging parameters:

  • Confocal microscopy provides optimal resolution for membrane proteins

  • Z-stack acquisition helps visualize the complete distribution pattern

What strategies are effective for studying SLITRK5 knockout phenotypes?

When investigating SLITRK5 knockout models:

• Generation approaches:

  • Conventional knockout mice have been created and characterized

  • CRISPR/Cas9 can be used for tissue-specific or inducible knockout systems

• Phenotypic assessment:

  • Neuronal phenotypes:

    • Assess neurite outgrowth in primary cultured neurons

    • Evaluate synapse formation and morphology

    • Examine obsessive-compulsive-like behaviors in animal models

  • Bone phenotypes:

    • Measure alkaline phosphatase (ALP) activity

    • Perform alizarin red staining to assess mineralization

    • Analyze expression of osteoblast marker genes (Runx2, Sp7, Bsp, Ocn, Alpl)

• Molecular analysis:

  • Measure levels of Hh target genes (Gli1, Gli2, Ptch1) to assess pathway activity

  • Examine protein levels of glutamate receptor subunits in neural tissues

  • Use PSD-enriched fractions of synaptosomes for detailed synaptic analysis

• Rescue experiments:

  • Re-express wild-type or mutant SLITRK5 in knockout backgrounds

  • Use domain-specific constructs to map functional regions

  • Employ pharmacological modulators of associated pathways (Hh pathway, BDNF/TrkB)

Why might I observe different molecular weights for SLITRK5 in Western blots?

Variations in SLITRK5's apparent molecular weight on Western blots occur due to several factors:

• Post-translational modifications:

  • Glycosylation significantly increases the observed molecular weight

  • SLITRK5 undergoes extensive glycosylation as a membrane protein

  • The predicted size is 107 kDa, but observed sizes range from 100-160 kDa

• Protein isoforms:

  • Up to 2 different isoforms have been reported for SLITRK5

  • These isoforms may display different electrophoretic mobility

• Sample preparation:

  • Insufficient denaturation can affect migration

  • Complete reduction of disulfide bonds is important for accurate sizing

  • Heat samples at 95°C for 5 minutes in Laemmli buffer with reducing agents

• Gel concentration:

  • Lower percentage gels (6-8%) provide better resolution for high molecular weight proteins

  • Consider gradient gels (4-15%) for improved separation

To confirm antibody specificity, use blocking peptides or SLITRK5-knockout samples as controls .

How can I improve signal-to-noise ratio when using SLITRK5 antibodies?

To enhance signal specificity and reduce background:

• Optimization strategies for Western blotting:

  • Increase blocking time and concentration (5% milk or BSA, minimum 1 hour)

  • Use longer washing steps (5 x 5 minutes with TBS-T)

  • Titrate primary antibody concentration (start with 1:1000 and adjust)

  • Consider overnight incubation at 4°C for primary antibody

  • Use highly-specific secondary antibodies with minimal cross-reactivity

• For immunofluorescence/immunohistochemistry:

  • Include 0.1-0.3% Triton X-100 for membrane protein accessibility

  • Extend blocking time to 2 hours at room temperature

  • Consider using specialized blocking reagents for neural tissues

  • Employ tyramide signal amplification for weak signals

  • Use higher antibody concentrations for fixed tissues (1:200) versus cell cultures (1:500)

• Validation approaches:

  • Compare results from multiple antibodies targeting different epitopes

  • Include appropriate negative controls (blocking peptides, knockout samples)

  • Consider preabsorption tests to confirm specificity

What are the considerations for studying SLITRK5 in different species models?

When investigating SLITRK5 across species:

• Conservation analysis:

  • Human SLITRK5 shares 97% amino acid identity with mouse and 98% with canine SLITRK5

  • SLITRK5 gene orthologs have been reported in mouse, rat, bovine, frog, chimpanzee, and chicken

• Antibody cross-reactivity:

  • Verify species reactivity for each antibody

  • Some antibodies are specifically validated for human and mouse

  • Others show broader reactivity including rat, dog, cow, sheep, pig, and horse

• Model-specific considerations:

  • Mouse models: Most extensively studied for both neuronal and bone phenotypes

  • Rat models: Useful for behavioral and neurological studies

  • Cell culture systems: Human cell lines (HEK293, Saos2) and mouse cell lines (C3H10t1/2, 3T3) have been successfully used

• Experimental readouts:

  • Account for species-specific differences in antibody recognition

  • Consider species-specific differences in signaling pathways and protein interactions

  • Validation of knockout phenotypes may vary between species

What are the emerging research areas for SLITRK5 beyond current applications?

Several promising research directions are developing:

• Therapeutic targeting for bone disorders:

  • As a negative regulator of hedgehog signaling in osteoblasts, SLITRK5 inhibition represents a potential therapeutic strategy

  • Development of specific inhibitors or neutralizing antibodies could enhance bone formation

  • Study of SLITRK5 in age-related bone loss and osteoporosis models

• Neuropsychiatric disorder connections:

  • SLITRK5 deficiency leads to obsessive-compulsive–like behaviors in mice

  • Further investigation of SLITRK5 variants in human neuropsychiatric conditions

  • Potential therapeutic targeting for OCD-spectrum disorders

• Cancer biology:

  • SLITRK5 is markedly upregulated in many neurological tumors

  • Investigation of its role in tumor cell growth, migration, and response to therapy

  • Potential diagnostic biomarker development

• Interaction with additional signaling pathways:

  • Beyond hedgehog and neurotrophin pathways, SLITRK5 may interact with other signaling mechanisms

  • Systematic interactome analysis could reveal novel functions

What methodological advances might improve SLITRK5 research?

Emerging technologies that could enhance SLITRK5 research include:

• Advanced imaging approaches:

  • Super-resolution microscopy for detailed subcellular localization

  • Live-cell imaging of SLITRK5 trafficking using fluorescent protein fusions

  • FRET/BRET approaches for real-time interaction studies

• Single-cell analysis:

  • Single-cell RNA-seq to identify cell-specific expression patterns

  • Single-cell proteomics for protein interaction studies

  • Spatial transcriptomics to map SLITRK5 expression in complex tissues

• Structural biology approaches:

  • Cryo-EM studies of SLITRK5 in complex with binding partners

  • Detailed mapping of interaction interfaces

  • Structure-based design of specific modulators

• In vivo models:

  • Cell-type specific conditional knockout models

  • Transgenic reporter systems for real-time visualization

  • Humanized mouse models for translational studies

These methodological advances will help address current knowledge gaps and accelerate progress in understanding SLITRK5 biology and its therapeutic potential.