CELSR3 Antibody

What is CELSR3 and what cellular functions does it regulate?

CELSR3 is part of the adhesion-class G protein-coupled receptors family. It contains large extracellular domains that undergo autoproteolytic cleavage at a conserved GPS within the GAIN domain. CELSR3 has been shown to regulate neural precursor cell fate decisions through the Wnt signaling pathway and plays important roles in:

• Axonogenesis and neuron migration in the developing brain

• Cell-cell adhesion mechanisms

• Glutamatergic synapse formation and maintenance

• Neuroblast migration in postnatal brain development

Research has demonstrated that CELSR3 knockout results in approximately 24% fewer presynaptic puncta, 36% fewer postsynaptic puncta, and 38% fewer colocalized puncta characteristic of glutamatergic synapses, indicating its crucial role in synapse formation .

How should I select the appropriate CELSR3 antibody for my specific application?

When selecting a CELSR3 antibody, consider these application-specific factors:

• For Western Blot: Select antibodies validated for detecting specific bands at appropriate molecular weights (approximately 260 kDa and 400 kDa for CELSR3)

• For Flow Cytometry: Choose fluorophore-conjugated antibodies (such as PE-conjugated) or primary antibodies compatible with secondary detection systems

• For Immunostaining: Verify that the antibody has been validated in fixed tissue or cells with appropriate controls

Always review the technical literature for each antibody to confirm it targets the specific region of CELSR3 relevant to your research. For example, some antibodies target the GAIN region, which is essential for studies involving T-cell redirection therapeutics .

What are the recommended protocols for CELSR3 antibody validation?

Proper antibody validation should include:

• Positive controls: Use cell lines known to express CELSR3 (e.g., SH-SY5Y neuroblastoma cells or HEK293 cells transfected with human CELSR3)

• Negative controls: Include isotype control antibodies (e.g., MAB0041) and CELSR3-negative cell lines (e.g., DU145)

• Knockout validation: Compare antibody performance in wild-type versus CELSR3 knockout models

• Cross-reactivity testing: If studying multiple species, confirm cross-reactivity (as demonstrated with human and mouse cell lines)

For flow cytometry specifically, validate using paired samples with:

• Target samples stained with CELSR3-specific antibody

• Control samples stained with isotype-matched control antibody

What are the optimal conditions for detecting CELSR3 in Western blot experiments?

Based on published protocols, the following conditions are recommended:

• Sample preparation: Prepare cell lysates using standard lysis buffers compatible with membrane proteins

• Gel conditions: Run samples under reducing conditions using Immunoblot Buffer Group 8

• Membrane: Use PVDF membrane for optimal protein binding

• Antibody concentration: Start with 0.1 μg/mL of anti-CELSR3 antibody (e.g., MAB7278)

• Secondary antibody: Use HRP-conjugated anti-mouse IgG (e.g., HAF007)

• Expected bands: Look for specific bands at approximately 260 kDa and 400 kDa

When analyzing results, be aware that the higher molecular weight band represents the full-length protein, while the lower molecular weight band may represent processed forms following autoproteolytic cleavage at the GPS domain.

How can I optimize CELSR3 detection using flow cytometry?

For optimal flow cytometry detection of CELSR3:

• Cell preparation: Use gentle dissociation methods to preserve membrane integrity

• Antibody concentration: Use approximately 10 μL of PE-conjugated antibody per 10^6 cells

• Cell types: SH-SY5Y neuroblastoma cells serve as positive controls for human CELSR3, while bEnd.3 mouse endothelioma cells can be used for mouse studies

• Controls: Include isotype control antibodies (e.g., IC0041P for PE-conjugated antibodies)

• Staining protocol: Follow standard membrane-associated protein staining protocols

Researchers should be aware that receptor density affects detection sensitivity. According to research findings, approximately 4,000 receptors per cell provide robust detection, while levels around 2,000 receptors per cell may give weaker signals .

What methods are effective for CELSR3 knockdown/knockout in experimental models?

Two validated approaches for CELSR3 manipulation include:

• shRNA knockdown:

  • Target the 3' untranslated region of CELSR3

  • Use lentiviral transduction systems with appropriate selection

  • Include scrambled sequence controls

  • Verify knockdown efficiency via Western blot using anti-CELSR3 antibody (1:1,000, NOVUS #NBP238975)

• CRISPR/Cas9 knockout:

  • Use multiple independent sgRNAs targeting CELSR3 (e.g., sgCELSR3_1, sgCELSR3_4, sgCELSR3_5)

  • Include sgGFP as negative control

  • Verify knockout efficiency through Western blot and functional assays

Following genetic manipulation, functional consequences can be assessed through cell proliferation assays, migration assays, and expression analysis of NEPC markers such as CHGA and SYP .

How can I use CELSR3 antibodies to investigate its role in cancer development?

CELSR3 has emerged as a potential biomarker and therapeutic target in several cancers, particularly neuroendocrine prostate cancer (NEPC). Researchers can:

• Analyze expression patterns: Compare CELSR3 expression across cancer subtypes and normal tissues using antibody-based techniques

  • Immunohistochemistry of tissue microarrays

  • Western blot of patient-derived samples

  • Flow cytometry of cancer cell lines

• Correlate with clinical outcomes: Investigate CELSR3 as a prognostic marker

  • CELSR3 gene expression has been linked to pN-stage and pM-stage in some cancers

  • High CELSR3 expression correlates with prognosis in some cancer types

• Functional studies: Use CELSR3 antibodies alongside genetic manipulation to assess phenotypic changes

  • CELSR3 knockdown has been shown to reduce NEPC tumor cell proliferation and migration

  • Effects on NEPC markers like CHGA and SYP can be monitored following manipulation

What is the methodology for using CELSR3 antibodies in T-cell redirection therapeutic research?

Research into CELSR3 as a therapeutic target involves:

• Antibody generation campaign:

  • Generate binders to the GAIN region of CELSR3 protein

  • Triage hits using biophysical and cellular assays

• Bispecific antibody construction:

  • Express potent binders as bispecific monoclonal antibodies (bs-mAb)

  • Engineer antibodies to simultaneously bind CELSR3 on tumor cells and CD3 receptor on T cells

• Efficacy testing:

  • Add CELSR3xCD3 bs-mAb and purified human pan T cells to CELSR3+ cell lines

  • Measure cell lysis (e.g., 83% maximum cell lysis in TCCSUP cells, 19% in PM154 cells)

  • Include CELSR3-negative cell lines (e.g., DU145) as controls

• Receptor density correlation:

  • Quantify CELSR3 surface expression by flow cytometry

  • TCCSUP: approximately 4,000 receptors per cell

  • PM154: approximately 2,000 receptors per cell

  • Determine threshold receptor density required for effective T-cell mediated cytolysis

How can I analyze the relationship between CELSR3 and glutamatergic synapse formation?

To investigate CELSR3's role in synapse formation:

• Comparative immunostaining:

  • Compare wild-type and CELSR3 knockout cultures

  • Use vGlut1 antibody for presynaptic puncta

  • Use PSD-95 antibody for postsynaptic puncta

  • Quantify colocalized puncta to identify glutamatergic synapses

• Electrophysiological assessment:

  • Record miniature excitatory postsynaptic currents (mEPSCs) in the presence of TTX (1 mM) and gabazine (10 μM)

  • Compare frequency, amplitude, and decay time constants between wild-type and CELSR3 knockout neurons

  • Note that loss of CELSR3 causes approximately 45% reduction in mEPSC frequency

• Molecular interaction studies:

  • Investigate CELSR3's relationship with other synaptic proteins

  • Compare with other adhesion molecules like Vangl2, which has opposing roles (Celsr3 promotes assembly whereas Vangl2 inhibits assembly)

What are common challenges when working with CELSR3 antibodies and how can they be addressed?

Researchers may encounter these challenges:

• Limited commercial antibody availability:

  • Researchers have noted that "there is no commercially available antibody that would allow reliable validation of CELSR3 protein expression in patient samples"

  • Solution: Generate custom antibodies or validate available options extensively with positive and negative controls

• Complex protein structure affecting detection:

  • CELSR3 undergoes autoproteolytic cleavage, resulting in multiple fragments

  • Solution: Target specific domains (e.g., GAIN region) and be aware of which fragment your antibody detects

• Variable expression levels:

  • CELSR3 expression may be low in some contexts, requiring sensitive detection methods

  • Solution: Optimize antibody concentration and detection systems; consider receptor density (threshold around 2,000-4,000 receptors per cell for robust detection)

How should I interpret variations in CELSR3 detection across different experimental systems?

When interpreting CELSR3 data across systems:

• Cell type considerations:

  • Expression varies significantly between neural, cancer, and normal tissues

  • CELSR3 is enriched in NEPC but not in normal tissues

  • Compare expression using standardized methods like RT-PCR with multiple Taqman probes

• Experimental context effects:

  • "Receptor expression of prostate tumor specific antigens has been shown to increase in more physiologically-relevant contexts, such as spheroids and xenograft models"

  • Consider evaluating expression in 2D cultures versus 3D models and in vivo systems

• Technical variation:

  • Establish quantitative benchmarks (e.g., receptors per cell by flow cytometry)

  • Use consistent protocols and antibody lots when comparing across experiments

  • Include appropriate controls in every experiment

What are the latest approaches for studying CELSR3 in cancer immunotherapy?

Recent advances in CELSR3 cancer immunotherapy research include:

• T-cell redirection therapeutics:

  • Development of bispecific antibodies targeting CELSR3 and CD3

  • Evaluation of cytolysis efficiency based on receptor density

  • Optimization of antibody binding to improve T-cell effector function activation at lower CELSR3 expression levels

• Target validation approaches:

  • Multi-step bioinformatics pipelines to identify NEPC-specific, overexpressed gene transcripts encoding cell surface proteins

  • Laser capture microdissection followed by RNA-seq to validate causal roles in NEPC

  • Comparative expression analysis across tissue types using GTEx and targeted RT-PCR with multiple probes

• Safety profiling:

  • Assessment of off-target activity against CELSR3-negative cell lines

  • Evaluation of CELSR3 expression in normal adult tissues to predict potential toxicity

  • Development of more sensitive detection methods to identify low-level expression in healthy tissues

How might CELSR3 research evolve to address current limitations in cancer diagnosis and treatment?

Future directions for CELSR3 research may include:

• Improved detection methods:

  • Development of more specific antibodies targeting different CELSR3 domains

  • Creation of multiplexed detection systems to simultaneously assess CELSR3 and related biomarkers

  • Integration with other emerging biomarkers like CEACAM5 and HMMR

• Therapeutic optimization:

  • Engineering of optimized CELSR3xCD3 bs-mAbs to increase cytolysis even at lower CELSR3 surface expression levels

  • Evaluation in more physiologically-relevant contexts like spheroids and xenograft models

  • Combination approaches with other treatment modalities

• Expanded understanding of biological mechanisms:

  • Further exploration of CELSR3's role in EMT and neuroendocrine differentiation

  • Investigation of upstream regulators and downstream effectors of CELSR3 signaling

  • Development of conditional knockout models to understand tissue-specific functions