wnt5b Antibody

What is WNT5B and how does it differ functionally from WNT5A?

WNT5B is a protein belonging to the WNT family with a molecular weight of approximately 40.3-42 kilodaltons. Despite sharing 83% amino acid identity with WNT5A, WNT5B often exhibits unique expression patterns and functional activities . For example:

• In hematopoiesis, WNT5B has divergent effects on IL-3 and GM-CSF-induced myeloid differentiation, while WNT5A does not affect these processes

• In pancreatic β-cells, WNT5B increases levels of NKX6.1 (a transcription factor controlling β-cell fate) when administered alone, whereas WNT5A alone does not have this effect

• In mammary cells, WNT5B demonstrates transforming capabilities, whereas WNT5A fails to induce transformation

Methodological approach: When studying WNT5B in relation to WNT5A, researchers should employ parallel experiments with both proteins and use specific antibodies that can distinguish between these closely related proteins. Western blotting followed by densitometry analysis can quantify expression levels in different tissues or under different conditions.

What are the main applications of WNT5B antibodies in research?

Based on commercial antibody information and research literature, WNT5B antibodies are commonly used in:

Methodological consideration: When selecting a WNT5B antibody, researchers should verify the epitope region to ensure specificity. Some antibodies (like those raised against amino acids 130-180) may detect both WNT5A and WNT5B due to sequence homology . Consider validating with recombinant proteins or knockout controls.

What signaling pathways does WNT5B activate or inhibit?

WNT5B primarily signals through:

• Non-canonical β-catenin-independent pathways:

  • JNK/c-JUN pathway (evidenced by elevated c-JUN levels in pancreatic β-cells)

  • PCP (planar cell polarity) pathway

  • Calcium-dependent pathways

• Canonical WNT signaling:

  • Often functions as an antagonist of canonical WNT signaling

  • In some contexts (e.g., cardiac development), WNT5B can activate canonical pathways

Methodological approach: To study WNT5B signaling, researchers should employ reporter assays like TCF/LEF luciferase reporter for canonical pathway activation and monitor phosphorylation status of JNK/c-JUN for non-canonical pathway analysis. Western blotting for downstream effectors can help delineate specific pathway activation.

How does WNT5B contribute to cancer progression and what are the implications for therapeutic targeting?

WNT5B has been implicated in several cancer types, particularly in breast cancer:

• Breast cancer: WNT5B is overexpressed in Triple Negative/Basal-Like Breast Cancer (TNBC/BLBC) and correlates with worse prognosis

  • Mechanistically, WNT5B promotes TAZ activation by upregulating SLUG transcription

  • Knockdown of WNT5B inhibits proliferation, migration, and invasion of breast cancer cells

  • WNT5B amplification occurs in 3.2% of breast cancers

• Therapeutic strategies:

  • WNT inhibitors like pyrvinium pamoate (CK1α activator) and LGK-974 (Porcupine inhibitor) reduce tumor growth in vivo

  • CK1α is expressed at low levels in BLBC, and its activation by pyrvinium represents a potential alternative treatment strategy

Methodological approach: Researchers investigating WNT5B as a therapeutic target should employ gene knockdown (siRNA, shRNA) or CRISPR-Cas9 approaches combined with both in vitro functional assays (proliferation, migration, invasion) and in vivo xenograft models. Patient-derived xenografts would provide more clinically relevant models for testing WNT5B-targeted therapies.

What are the experimental considerations when studying WNT5B's divergent effects on hematopoiesis?

WNT5B exhibits context-dependent effects on hematopoietic stem cells (HSCs) and progenitors:

Methodological considerations:

• Concentration matters: Effects with GM-CSF are observed at lower WNT5B concentrations (50 ng/mL) compared to effects with IL-3 (200 ng/mL)

• Timepoint analysis: Monitor changes in colony formation across multiple timepoints (initial decrease followed by increase with IL-3)

• Markers to assess:

  • Cell cycle markers: Cdk1, Cyclin D1

  • Primitive cell markers: GATA-2, Ifitm-1

  • Lineage markers: Lin+/Lin- ratio

• Functional assays: Colony formation unit (CFU) assay with specific subtypes (GM-CFU, M-CFU, G-CFU)

Research pitfall: Failing to account for potentially different receptor populations activated at different WNT5B concentrations could lead to inconsistent results.

How do WNT5B antibodies perform in detecting various WNT5B isoforms and how can specificity be validated?

WNT5B has at least two known isoforms, and appropriate validation is critical:

Detection considerations:

• Most commercial antibodies detect both isoforms of WNT5B

• The calculated molecular weight of WNT5B is ~40.3 kDa, but the observed weight in Western blots may be ~68 kDa due to post-translational modifications

Validation strategies:

• Positive controls: Use recombinant WNT5B proteins of known isoforms

• Negative controls:

  • WNT5B knockout/knockdown cells or tissues

  • Peptide competition assays with the immunizing peptide

• Cross-reactivity testing: Test against closely related proteins, particularly WNT5A

• Multi-antibody approach: Use antibodies targeting different epitopes to confirm results

Technical note: When selecting antibodies, examine the immunogen information. Antibodies raised against peptides within amino acids 130-180 of WNT5B may detect both WNT5A and WNT5B due to high sequence homology in this region .

What is the role of WNT5B in lymphatic vessels and how does this impact tumor metastasis?

WNT5B plays crucial roles in lymphatic vessel development and tumor lymphangiogenesis:

• Lymphatic development:

  • WNT5B is necessary and sufficient for lymphatic endothelial specification

  • Controls the transition of angioblasts to lymphatic progenitor cells in zebrafish

  • This function appears evolutionarily conserved from zebrafish to human embryonic stem cells

• Tumor lymphangiogenesis and metastasis:

  • WNT5B is secreted by tumor cells (e.g., melanoma) to induce tumor lymphangiogenesis

  • Enhances lymph vessel formation, permeability, and migration of lymphatic endothelial cells

  • Functions in a bi-directional pro-metastatic crosstalk between melanoma and lymphatic endothelial cells through a DLL4-Notch3-WNT5B axis

  • Decreases beta-catenin and ZO-1 expression on lymphatic endothelial cell membranes, reducing barrier function

Methodological approaches:

• 3D spheroid sprouting assays to measure lymphangiogenic potential

• Transwell permeability assays to assess lymphatic vessel integrity

• Co-culture systems with tumor cells and lymphatic endothelial cells to study bidirectional signaling

• In vivo metastasis models with fluorescently labeled tumor cells to visualize lymphatic invasion

How can researchers investigate the intersection of WNT5B with other signaling pathways in development and disease?

WNT5B intersects with several key signaling pathways, providing opportunities for mechanistic studies:

• Notch signaling:

  • The DLL4-Notch3-WNT5B axis mediates melanoma-lymphatic endothelial cell interactions

  • DLL4-Fc coating induces approximately four-fold increase in WNT5B expression in melanoma cells

• FGF signaling in development

• JAK/STAT, Ras/ERK, PI3K/PKB pathways:

  • WNT5B can modulate these pathways that are also activated by IL-3 and GM-CSF receptors

• TAZ/Hippo pathway in breast cancer:

  • WNT5B promotes TAZ activation via SLUG upregulation

Experimental approaches:

• Pathway inhibitor studies: Use specific inhibitors of each pathway to identify where WNT5B effects are blocked

• Protein-protein interaction studies: Co-immunoprecipitation, proximity ligation assays

• Transcriptional profiling: RNA-seq following WNT5B stimulation with/without inhibition of other pathways

• Receptor studies: Analysis of Frizzled receptor expression and function (note that while FZD8 has highest binding affinity to WNT5B in silico, knockdown studies suggest involvement of multiple receptors)

Research challenge: The context-dependent effects of WNT5B require careful experimental design with appropriate positive and negative controls for each signaling pathway being investigated.

What are the optimal conditions for using WNT5B antibodies in different applications?

Based on commercial antibody information and research practices:

Methodological notes:

• For IHC applications, formalin-fixed paraffin-embedded tissues require proper antigen retrieval as demonstrated in prostate tissue samples

• For Western blot, be aware that the observed molecular weight (~68 kDa) may differ from the calculated weight (~40.3 kDa) due to post-translational modifications

• Store WNT5B antibodies at 4°C for up to three months or at -20°C for up to one year for optimal stability

How can researchers distinguish between non-specific binding and true WNT5B signal?

To ensure specific detection of WNT5B:

• Peptide competition assay: Pre-incubate the WNT5B antibody with excess immunizing peptide before application to samples. Specific signals should be abolished or significantly reduced.

• Multiple antibody approach: Use antibodies targeting different epitopes of WNT5B. True signals should be consistent across antibodies.

• Genetic knockdown/knockout controls: Include WNT5B-depleted samples as negative controls.

• Cross-reactivity panel: Test antibody against recombinant WNT5A and other closely related WNT family proteins.

• Signal validation across applications: Confirm WNT5B expression using complementary techniques (e.g., verify IHC findings with Western blot).

Technical insight: If studying both WNT5A and WNT5B, select antibodies targeting non-homologous regions to avoid cross-reactivity. The central region (amino acids 130-180) has higher homology and antibodies against this region may detect both proteins .

What methodological approaches are best for studying WNT5B in cancer research?

For investigating WNT5B in cancer contexts, consider these specialized approaches:

• Patient stratification:

  • Analyze WNT5B expression in tumor subtypes (e.g., high expression in TNBC/BLBC)

  • Correlate with clinical outcomes and treatment responses

• Functional studies:

  • Knockdown/overexpression in appropriate cancer cell lines

  • Measure effects on hallmark cancer behaviors (proliferation, migration, invasion, colony formation)

  • Xenograft models to assess in vivo tumor growth and metastasis

• Mechanistic investigations:

  • Analyze WNT5B effects on TAZ activation and SLUG expression in breast cancer

  • Measure WNT pathway activation using reporter assays

  • Investigate drug sensitivity with WNT inhibitors (e.g., LGK-974, pyrvinium)

• Therapeutic potential assessment:

  • Combined inhibition of WNT5B with standard chemotherapies

  • Testing CK1α activation as a strategy in BLBC where CK1α is expressed at low levels