wnt11 Antibody

What is Wnt11 and what are its key functions in cellular signaling?

Wnt11 is a member of the Wnt protein family that functions as a ligand for frizzled family seven transmembrane receptors. In humans, the canonical Wnt11 protein is 354 amino acids with a molecular weight of approximately 39.2 kDa . Unlike canonical Wnt proteins that signal through β-catenin, Wnt11 predominantly activates non-canonical pathways.

Research demonstrates that Wnt11 plays crucial roles in:

• Development, regulating discrete tissue regions, typically signaling over short distances

• Cellular proliferation and transformation through β-catenin-independent mechanisms

• Migration and cytoskeletal rearrangement in intestinal epithelial cells

• Tumor microenvironment modulation, affecting immune cell function

• Matrix metalloproteinase (MMP) regulation, influencing cell invasion

Studies using intestinal epithelial cell models have established that Wnt11 can function in both autocrine and paracrine fashions to stimulate cellular proliferation . Recent evidence also implicates Wnt11 in complex developmental processes, with loss-of-function variants associated with situs inversus, cardiac defects, and renal hypodysplasia .

What are the main applications of Wnt11 antibodies in research?

Wnt11 antibodies serve as essential tools across multiple research applications:

Western Blotting: The most common application, detecting Wnt11 at approximately 39-40 kDa under reducing conditions. Under non-reducing conditions, dimeric forms can be observed . Optimal results typically employ 10% SDS-PAGE gels with 25-30μg total protein per lane .

Immunohistochemistry (IHC): Enables visualization of Wnt11 distribution in tissues, with documented expression in fetal lung, kidney, adult heart, liver, skeletal muscle, and pancreas . Paraffin-embedded sections typically require antigen retrieval and antibody dilutions ranging from 1:100-1:500 .

Immunocytochemistry/Immunofluorescence (ICC/IF): Allows subcellular localization studies, with Wnt11 typically showing cytoplasmic localization in cells such as LNCaP prostate cancer cells .

Functional Studies: Wnt11 antibodies enable:

• Depletion of Wnt11 from conditioned media (4 μg/ml) to confirm specificity of biological effects

• DNA-affinity precipitation assays (DAPA) to study transcriptional regulation of Wnt11 expression

• Neutralization of Wnt11 signaling in cell culture experiments

Detection in hypoxia studies: Wnt11 antibodies have proven valuable for tracking hypoxia-induced expression changes in various experimental models .

How does Wnt11 expression change in response to hypoxia?

Hypoxia consistently upregulates Wnt11 expression across multiple cell types through a well-defined molecular pathway. Experimental data demonstrates that hypoxic conditions (1% O₂) significantly increase Wnt11 mRNA and protein levels compared to normoxic conditions (21% O₂) .

This regulation occurs through the Von Hippel-Lindau (VHL)-Hypoxia-Inducible Factor (HIF) axis:

• Higher basal levels of Wnt11 protein are observed in Vhl-deleted cells (lenti-Cre infected Vhlf/f)

• Hypoxic mimetics including CoCl₂, DFO, and DMOG (0.1 mM) effectively induce Wnt11 expression

• HIF-1α is the predominant transcriptional regulator, as Hif-1α knockout substantially attenuates Wnt11 expression in response to hypoxia

• Inactivation of the Vhl gene results in increased Wnt11 mRNA in liver and duodenum models, with values normalized to Tbp mRNA showing significant upregulation (p < 0.05)

This relationship has clinical relevance in cancer treatment. In glioma models treated with bevacizumab (an antiangiogenic therapy, 6 mg/kg), treatment increases tumor hypoxia, leading to elevated HIF-1α/HIF-2α levels and corresponding increases in Wnt11 expression . The functional consequence includes enhanced MMP-2/MMP-9 activities promoting invasion and migration.

How does Wnt11 contribute to tumor immune evasion mechanisms?

Wnt11 functions as a key mediator of immune evasion in cancer microenvironments through multiple coordinated mechanisms:

Direct CD8⁺ T-cell suppression: Cancer cells expressing high Wnt11 levels directly inhibit CD8⁺ T-cell proliferation and cytotoxic function through repression of the transcription factor AFF3 . Knockdown of Wnt11 promotes T-cell-mediated cancer cell killing in co-culture experiments across multiple ratios of effector:target cells .

Chemokine modulation: Wnt11 suppresses expression and secretion of T-cell-attracting chemokines CXCL10 and CCL4. When Wnt11 is knocked down:

• mRNA expression and secretion of Cxcl10 and Ccl4 increase significantly

• This occurs through an AFF3-dependent mechanism

• Enhanced T-cell recruitment operates via CXCL10/CXCR3 and CCL4/CCR5 axes

Macrophage polarization: Wnt11 promotes accumulation of CD206⁺CD163⁺ suppressive macrophages. Flow cytometry analysis shows significantly decreased CD206⁺CD163⁺ macrophage counts in Wnt11 knockdown tumors .

In vivo evidence: Experimental data from multiple syngeneic mouse models (MC38, Panc02, CT26, KPC) demonstrates:

• Wnt11 knockdown significantly increases CD8⁺ T-cell infiltration

• Enhances expression of cytotoxic markers (GZMB, TNFα)

• Reduces tumor burden and improves survival in immunocompetent but not immunodeficient mice

• Sensitizes tumors to anti-PD-1 immunotherapy

These findings establish Wnt11 as a potential therapeutic target in combination with immune checkpoint inhibitors, particularly for liver metastases where Wnt11 expression correlates with poor prognosis.

What mechanisms regulate WNT11 gene expression?

WNT11 gene expression is regulated by multiple transcriptional and physiological mechanisms:

Hypoxia-Dependent Regulation:

• HIF-1α directly regulates WNT11 transcription during hypoxia

• Attenuated WNT11 expression occurs in Hif-1α KO cells exposed to hypoxia mimetics

• Overexpression of HIF-1α enhances WNT11 expression even under normoxic conditions

• This regulation involves the VHL pathway, as demonstrated by increased Wnt11 mRNA in Vhl-knockout tissues

Inflammatory Signaling Pathways:

• TNFα stimulation induces WNT11 expression in breast cancer cells

• This occurs through direct binding of Early Growth Response Protein 1 (EGR1) to the WNT11 promoter

• DNA-affinity precipitation assays (DAPA) confirm that EGR1 binding sites (EBS-1 and EBS-2) in the WNT11 promoter region (-22 to +18 nucleotides) bind EGR1 in response to TNFα stimulation

Tissue-Specific Expression Patterns:

• WNT11 is expressed in fetal lung, kidney, and in adult heart, liver, skeletal muscle, and pancreas

• Expression in intestinal tissue shows relatively uniform distribution between differentiated villus and surface epithelium, unlike some canonical Wnt ligands that show restricted expression patterns

Understanding these regulatory mechanisms provides opportunities to manipulate WNT11 expression therapeutically, particularly in contexts where its expression drives disease progression or therapy resistance.

How do C-terminal modifications affect Wnt11 protein function?

C-terminal domains of Wnt11 play crucial roles in determining protein stability, signaling capacity, and dimerization ability. Recent research comparing wild-type and C-terminally truncated Wnt11 variants demonstrates:

Protein Stability Effects:

• WNT11 c.814delG (a naturally occurring variant) shows reduced protein abundance compared to wild-type WNT11

• Similarly, WNT11 Δ850-1062 displays decreased protein levels in expression studies

• These reduced protein concentrations directly correlate with reduced functional potency in biological assays

Dimerization Capacity:

• Wild-type WNT11 readily forms protein dimers under non-reducing conditions

• WNT11 Δ814-1062, despite C-terminal truncation, maintains dimerization ability in vivo

• Dimerization appears to be necessary but insufficient for full signaling activity

Functional Consequences:

• The C-terminally truncated variant WNT11 Δ814-1062 demonstrates strong dominant-active signaling despite missing substantial C-terminal sequences

• This suggests specific C-terminal regions may have inhibitory functions in the native protein

• Different truncation points in the C-terminus result in dramatically different functional outcomes

Clinical Relevance:

• A homozygous human WNT11 loss-of-function variant (c.814delG) has been identified in a patient with situs inversus totalis, complex heart defects, and renal hypodysplasia

• This variant demonstrates the critical importance of proper Wnt11 function in human development

These findings highlight the complex structure-function relationships within the Wnt11 protein and suggest that targeted modifications of the C-terminal domain could potentially generate Wnt11 variants with enhanced or specialized signaling properties.

What are the optimal conditions for detecting Wnt11 by Western blot?

Successful Western blot detection of Wnt11 requires optimization of multiple parameters:

Sample Preparation:

• Cell/tissue lysis: RIPA buffer with protease inhibitor cocktail

• For secreted Wnt11: Concentrate conditioned media 20-fold using centrifugal filter units

• Protein loading: 25-30μg total protein per lane

• Include both reducing and non-reducing conditions to detect monomeric (~39kDa) and dimeric forms

Gel Electrophoresis Parameters:

• Gel percentage: 10% SDS-PAGE provides optimal resolution

• Positive controls:

  • Recombinant Wnt11

  • Lysates from known Wnt11-expressing cells (LNCaP, HEK-293T, A431)

• Negative controls: Lysates from Wnt11 knockdown cells

Transfer and Detection Protocol:

• Membrane: PVDF preferred for optimal protein retention

• Blocking: 5% non-fat milk or BSA in TBST

• Primary antibody: 1:1000 dilution at 4°C overnight (optimize for specific antibody)

• Secondary antibody: HRP-conjugated anti-rabbit or anti-goat IgG depending on primary species

• Detection system: Enhanced chemiluminescence with 30sec-5min exposure times

Special Considerations:

• For dimerization studies: Run parallel samples under reducing vs. non-reducing conditions

• To confirm specificity: Include Wnt11 knockdown samples

• For normalization: α-tubulin, lamin A/C, or actin depending on the cellular fraction

• For secreted Wnt11: Use Ponceau S staining for normalization

Using these conditions, Wnt11 typically appears as a distinct band at approximately 39-40kDa under reducing conditions, with potential additional bands at higher molecular weights under non-reducing conditions representing dimeric forms .

How can researchers generate and validate Wnt11-secreting cells for functional studies?

Establishing reliable Wnt11-secreting cells for functional studies requires careful consideration of expression systems and validation methods:

Expression System Options:

• Stable Transfection Approach:

  • Clone full-length mouse or human Wnt11 cDNA into an expression vector (e.g., pcDNA3)

  • Transfect target cells via electroporation (300V, 1 millifarad) or lipofection

  • Select with appropriate antibiotics (e.g., 1mg/ml G418)

  • Isolate and expand individual clones or stable populations

• Inducible Expression Systems:

  • Use tetracycline-regulated or other inducible promoter systems

  • Enables temporal control of Wnt11 secretion during experiments

Validation Methods:

• Protein Expression Verification:

  • Western blot of cell lysates using validated anti-Wnt11 antibodies

  • Expected band at approximately 39kDa under reducing conditions

  • Compare expression levels across multiple clones

• Secretion Confirmation:

  • Collect conditioned media after 2-3 days of culture in serum-free conditions

  • Concentrate media by centrifugation or filtration

  • Western blot analysis of concentrated media

  • Quantify secreted Wnt11 by ELISA if available

• Functional Validation:

  • Conditioned media effects on known Wnt11-responsive cells

  • Neutralization experiments using Wnt11 antibodies (4μg/ml)

  • Comparison with recombinant Wnt11 protein standards

Experimental Applications:

• Direct Co-culture Studies:

  • Seed Wnt11-secreting cells together with target cells

  • Monitor proliferation, migration, or transformation effects

• Indirect Co-culture:

  • Use transwell systems with Wnt11-secreting cells in upper chamber

  • Enables study of paracrine effects without direct cell contact

• Conditioned Media Applications:

  • Treatment of target cells with Wnt11-containing media

  • Concentration-response studies to determine potency

  • Comparison with control (vector-transfected) conditioned media

Using these approaches, researchers can establish robust cellular systems for studying both autocrine and paracrine Wnt11 signaling effects, as demonstrated in studies examining Wnt11's impact on intestinal epithelial cell proliferation and transformation .

What methods are available for studying Wnt11 interactions with the tumor immune microenvironment?

Investigating Wnt11's role in tumor-immune interactions requires specialized methodologies spanning in vitro, ex vivo, and in vivo approaches:

In Vitro Co-culture Systems:

• T-cell Proliferation Assays:

  • Co-culture of Wnt11-expressing or knockdown cancer cells with CD8⁺ T-cells

  • Measurement of T-cell proliferation via CFSE dilution or BrdU incorporation

  • Quantification of proliferation inhibition compared to control conditions

• Cytotoxicity Assays:

  • Antigen-specific killing assays using OVA-specific OT1 CD8⁺ T-cells and OVA-expressing cancer cells

  • Co-culture at multiple effector:target ratios

  • Comparison between control, Wnt11 knockdown, and rescue conditions

• Chemokine Expression Analysis:

  • Quantification of T-cell-attracting chemokines (CXCL10, CCL4) by qPCR and ELISA

  • Pathway analysis using receptor antagonists (CXCR3 and CCR5 inhibitors)

  • Rescue experiments with recombinant chemokines

Ex Vivo Analysis:

• Flow Cytometry Immunophenotyping:

  • Comprehensive immune cell profiling of tumor tissues

  • Quantification of CD8⁺ T-cell infiltration and activation markers

  • Analysis of suppressive cell populations (CD206⁺CD163⁺ macrophages)

• Multiplex Immunohistochemistry:

  • Simultaneous detection of multiple markers (CD8, I-A/I-E, CD206)

  • Spatial relationship analysis between tumor cells and immune populations

  • Quantification of marker co-expression patterns

In Vivo Models:

• Syngeneic Tumor Models:

  • Intrasplenic injection to establish liver metastasis models

  • Comparison between immunocompetent and immunodeficient mice

  • Measurement of tumor burden, survival, and immune infiltration

• Checkpoint Inhibitor Response Studies:

  • Treatment of Wnt11-expressing vs. knockdown tumors with anti-PD-1 antibodies

  • Assessment of therapeutic synergy between Wnt11 targeting and immunotherapy

  • Survival and tumor burden analysis

• Adoptive T-cell Transfer:

  • Transfer of labeled T-cells into tumor-bearing mice

  • Tracking T-cell infiltration, retention, and function in Wnt11-high vs. Wnt11-low tumors

Molecular Analysis Techniques:

• RNA Sequencing:

  • Transcriptional profiling of tumor and immune cell populations

  • Identification of differentially expressed genes in Wnt11-high vs. Wnt11-low conditions

  • Pathway analysis to identify key regulatory networks

• Protein Interaction Studies:

  • Immunoprecipitation to identify Wnt11-binding partners in immune cells

  • Receptor binding assays to characterize direct interactions

These methodologies provide a comprehensive toolkit for dissecting the complex roles of Wnt11 in tumor-immune interactions, offering insights into potential therapeutic targeting strategies.

Why might Wnt11 antibodies show inconsistent results across applications?

Inconsistent Wnt11 detection across research applications can stem from multiple factors related to protein characteristics, antibody properties, and experimental conditions:

Protein-Related Factors:

• Post-translational modifications:

  • Wnt11 undergoes glycosylation affecting epitope recognition

  • C-terminal truncations significantly impact protein stability and detection

  • Compare reducing vs. non-reducing conditions to assess structure effects

• Expression levels:

  • Endogenous Wnt11 is often expressed at low levels

  • Hypoxia significantly upregulates expression, potentially leading to variability between normoxic/hypoxic conditions

  • Consider concentration methods for low-abundance samples

Antibody-Related Factors:

• Epitope specificity:

  • Antibodies recognizing different Wnt11 regions yield varying results

  • Some epitopes may be masked by protein conformation or interactions

  • Test multiple antibodies targeting different epitopes (N-terminal vs. middle region vs. C-terminal)

• Cross-reactivity:

  • Potential cross-reactivity with other Wnt family members

  • Species-specific variations in epitope sequences

  • Validate using Wnt11 knockdown samples as negative controls

Application-Specific Considerations:

• Western blot inconsistencies:

  • Transfer efficiency issues due to Wnt11's hydrophobicity

  • Protein dimerization affecting migration patterns

  • Solution: Optimize transfer conditions; include non-reducing samples

• Immunohistochemistry variations:

  • Fixation effects on epitope accessibility

  • Antigen retrieval requirements differ between tissues

  • Solution: Compare multiple antigen retrieval methods; optimize antibody dilutions (1:100-1:500)

• Flow cytometry challenges:

  • Cell permeabilization requirements for intracellular detection

  • Competition with binding partners in native state

  • Solution: Test different permeabilization protocols; include blocking steps

Validation Strategy:
To address inconsistencies, implement a multi-faceted validation approach:

• Compare multiple antibodies in parallel experiments

• Include positive controls (recombinant Wnt11, overexpression models)

• Use negative controls (Wnt11 knockdown, pre-absorption with recombinant protein)

• Document lot numbers and establish reference standards for cross-comparison

By systematically addressing these factors, researchers can significantly improve consistency in Wnt11 detection across diverse experimental platforms.

What factors affect the stability of Wnt11 protein in experimental conditions?

Wnt11 protein stability is influenced by multiple physical, chemical, and biological factors that must be carefully controlled:

Physical and Chemical Factors:

• Temperature effects:

  • Wnt proteins are highly temperature-sensitive

  • Maintain samples at 4°C during preparation

  • Avoid repeated freeze-thaw cycles

  • For long-term storage, maintain at -80°C

• pH sensitivity:

  • Optimal stability observed at pH 6.5-7.4

  • Buffer choice affects stability (phosphate buffers generally preferred)

  • Document pH conditions in experimental protocols

• Oxidation susceptibility:

  • Cysteine residues in Wnt11 are prone to oxidation

  • Include reducing agents (DTT, β-mercaptoethanol) in storage buffers

  • Minimize exposure to atmospheric oxygen

Biological Factors:

• Proteolytic degradation:

  • Wnt11 is susceptible to proteolytic cleavage

  • Include comprehensive protease inhibitor cocktails in all buffers

  • For conditioned media, add inhibitors immediately upon collection

• Binding protein interactions:

  • Carrier proteins (e.g., BSA) can enhance stability

  • Heparin or heparan sulfate can stabilize Wnt proteins

  • Consider adding 0.1-0.5% BSA to storage buffers

• C-terminal integrity:

  • C-terminal truncations significantly impact protein stability

  • WNT11 c.814delG and WNT11 Δ850-1062 show reduced stability

  • C-terminal regions may play critical roles in proper folding

Experimental Handling Best Practices:

• Collection of conditioned media:

  • Harvest at optimal timepoints (typically 24-72 hours post-seeding)

  • Centrifuge immediately to remove cell debris (3000g, 10 minutes)

  • Process immediately or flash-freeze in single-use aliquots

• Concentration methods:

  • Centrifugal filter units provide gentle concentration for secreted Wnt11

  • 20-fold concentration typically sufficient for detection

  • Validate recovery efficiency using spike-in experiments

• Storage conditions:

  • Store in small, single-use aliquots to avoid repeated freeze-thaw

  • Consider addition of 10-20% glycerol for cryoprotection

  • Document storage duration in experimental protocols

Implementation of standardized handling protocols addressing these factors is essential for cross-laboratory reproducibility in Wnt11 research and accurate interpretation of experimental results.

Common Wnt11 Antibody Applications and Recommended Conditions