DVL3 Antibody

What is DVL3 and why is it significant for research?

DVL3 (Dishevelled-3) is a multivalent scaffold protein that plays an essential role in Wnt signaling during development . As one of three Dishevelled family members in mammals, DVL3 is involved in both canonical and non-canonical Wnt pathways, making it a crucial target for studying developmental processes, cancer progression, and various cellular signaling mechanisms. Research has revealed that DVL3 forms supermolecular complexes ranging from homodimers to well-defined structures of 0.4-2.0 MDa, with complex formation being stimulated by Wnt3a signaling within 30 minutes of exposure . This dynamic complex assembly is vital for proper signal transduction in the Wnt pathway.

What are the common applications for DVL3 antibodies in research?

DVL3 antibodies are extensively utilized in multiple research applications:

These applications allow researchers to study DVL3 protein expression levels, subcellular localization, interaction partners, and post-translational modifications in various experimental contexts .

How do I select the appropriate DVL3 antibody for my experiment?

When selecting a DVL3 antibody for your experiment, several critical factors should be considered:

• Species reactivity: Confirm the antibody recognizes DVL3 from your experimental species. Available antibodies show reactivity with human, mouse, rat, hamster, and other species .

• Specificity: Verify whether the antibody is specific to DVL3 or cross-reacts with other Dishevelled family members. Some antibodies, like #3218, specifically detect DVL3 without cross-reacting with DVL2 .

• Application compatibility: Ensure the antibody is validated for your intended application. Check published literature and manufacturer data showing successful use in your application of interest .

• Clonality: Choose between polyclonal antibodies (offering broader epitope recognition) or monoclonal antibodies (providing higher specificity) .

• Target region: Consider whether your experiment requires detection of specific DVL3 domains or phosphorylation sites, especially for studies focused on Wnt signaling dynamics .

Review validation data, including western blot images and immunohistochemistry results, to confirm the antibody performs reliably in conditions similar to your experimental setup.

How should DVL3 antibodies be optimized for studying supermolecular complex formation?

To study DVL3 supermolecular complex formation effectively, implement the following methodological approach:

• Cell stimulation protocol: For studying Wnt-dependent complexes, stimulate cells with purified Wnt3a for specific time intervals (30 min, 1 hour) as this has been shown to induce progressive complex formation with detectable changes occurring within 30 minutes .

• Sample preparation: Use steric-exclusion chromatography to separate DVL3-based complexes by size. This technique has successfully resolved complexes ranging from homodimeric DVL3 to supermolecular complexes of 0.4-2.0 MDa .

• Immunoprecipitation optimization: For FLAG-tagged DVL3, use anti-FLAG immunoprecipitation followed by SDS-PAGE separation. For endogenous DVL3, use specialized antibodies at concentrations of 0.5-4.0 μg per 1.0-3.0 mg of total protein lysate .

• Detection sensitivity: Employ fluorescence correlation microscopy to characterize the dynamic nature of DVL3 complexes in living cells, complementing biochemical approaches .

• Controls: Include DVL1/2/3 knockdown controls to distinguish specific effects, as studies have shown that knockdown of each Dvl isoform affects complex formation differently. Notably, Dvl3 knockdown precludes complex formation, while Dvl1 knockdown specifically blocks Wnt3a-induced increases in complex size .

This multifaceted approach enables comprehensive analysis of DVL3's role in Wnt signaling complex assembly and dynamics.

What is the significance of DVL3 phosphorylation and how can it be analyzed using antibodies?

DVL3 phosphorylation represents a critical regulatory mechanism within Wnt signaling cascades. To effectively analyze DVL3 phosphorylation:

• Experimental design: Express FLAG-DVL3 in HEK293 cells with or without specific kinases of interest, followed by immunoprecipitation using anti-FLAG antibodies .

• Phosphorylation analysis techniques:

  • Western blotting with phospho-specific antibodies

  • Mobility shift assays (DVL3 appears as 88-93 kDa bands with characteristic shifts when phosphorylated)

  • Mass spectrometry for comprehensive phosphorylation mapping

• Kinase modulation: Employ specific kinase inhibitors such as PF-670462 (CK1ε inhibitor at 10 μM) to evaluate the contribution of specific kinases to DVL3 phosphorylation patterns .

• Functional correlation: Combine phosphorylation analysis with functional assays such as the TopFlash reporter assay in DVL1/2/3-null HEK293 T-REx cells transfected with DVL3 variants to connect phosphorylation status with Wnt signaling activation .

• FlAsH labeling approach: For live-cell imaging of DVL3 conformational changes associated with phosphorylation, utilize the DVL3 FlAsH III sensor methodology with 500 nM FlAsH and 12.5 μM 1,2-ethanedithiol (EDT) as described in previous protocols .

This comprehensive approach links DVL3's phosphorylation state to its functional role in Wnt signaling cascades.

How can subcellular localization of DVL3 be accurately determined using immunofluorescence?

For precise subcellular localization analysis of DVL3:

• Sample preparation protocol:

  • Grow cells on appropriate culture dishes (24-well plates for ICC)

  • Fix with 4% paraformaldehyde followed by permeabilization with 0.1-0.5% Triton X-100

  • Block with 5% BSA or appropriate serum

• Antibody dilution optimization: For immunofluorescence/ICC applications, use DVL3 antibody at 1:50-1:500 dilution, with titration recommended for each experimental system .

• Co-staining strategy: Implement co-staining with markers for specific subcellular compartments (nuclear, cytoplasmic, membrane, endosomal) to precisely define DVL3 localization under different signaling conditions.

• Quantitative analysis approach: Apply semi-quantitative evaluation methods similar to those used in glioblastoma studies, where both signal intensity and subcellular distribution patterns are systematically quantified .

• Physiological interpretation: Consider that DVL3 has been observed predominantly in the cytoplasm (97% of glioblastoma samples), with 44% of samples showing co-expression in the nucleus. Strong expression levels correlate significantly with nuclear localization (P = 6.33 × 10^-5) .

This methodology enables detection of key subcellular translocation events associated with DVL3's role in Wnt signaling activation.

How can non-specific binding be minimized when using DVL3 antibodies?

To reduce non-specific binding in DVL3 antibody applications:

• Western blotting optimization:

  • Increase blocking stringency using 5% non-fat dry milk or BSA in TBST

  • Optimize primary antibody dilution within the 1:2000-1:6000 range

  • Extend washing steps (5 × 5 minutes with TBST)

  • Use antibodies specifically validated not to cross-react with other Dishevelled family members

• Immunoprecipitation refinement:

  • Pre-clear lysates with protein A/G beads before adding DVL3 antibody

  • Use the precise recommended antibody amount (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Include appropriate negative controls (non-specific IgG, lysates from DVL3-knockout cells)

• Immunohistochemistry considerations:

  • Optimize antigen retrieval methods; studies indicate optimal results with TE buffer pH 9.0, with citrate buffer pH 6.0 as an alternative

  • Titrate antibody concentration within 1:500-1:2000 range

  • Include absorption controls with blocking peptides when available

• Fluorescence labeling specificity:

  • For FlAsH labeling techniques, incorporate the EDT wash step (250 μM EDT) to reduce non-specific labeling as specified in published protocols

  • Use appropriate negative controls for each experimental system

These methodological refinements significantly enhance signal-to-noise ratio and experimental reliability.

What are the critical factors affecting DVL3 antibody performance in different experimental conditions?

Several factors critically influence DVL3 antibody performance:

• Buffer composition effects:

  • Storage buffer stability: DVL3 antibodies typically require storage in PBS with 0.02% sodium azide and 50% glycerol at pH 7.3

  • Temperature sensitivity: Store at -20°C, where stability is maintained for one year after shipment without requiring aliquoting

• Sample preparation considerations:

  • Phosphorylation state: Since DVL3 undergoes extensive phosphorylation, sample handling should preserve phosphorylation status if relevant to the experiment

  • Complex integrity: For studies of supermolecular complexes, gentle lysis conditions are essential to maintain complex architecture

• Epitope accessibility variables:

  • Fixation effects: Over-fixation can mask epitopes, particularly for antibodies targeting conformational epitopes

  • Protein interactions: DVL3's involvement in large protein complexes may obscure antibody binding sites in native conditions

• Detection system compatibility:

  • Signal amplification requirements vary based on expression level, with endogenous DVL3 detection often requiring more sensitive detection systems

  • The observed molecular weight can range from 78 kDa (calculated) to 88-93 kDa due to post-translational modifications

• Cell/tissue type variability:

  • DVL3 antibody performance has been successfully validated in multiple cell types including A549, MCF-7, Raji, and HeLa cells

  • Tissue-specific considerations include optimizing antigen retrieval for different tissue types as demonstrated in colon, prostate, and cervical cancer tissues

Understanding these variables enables strategic optimization of experimental protocols for specific research questions.

How do you validate DVL3 antibody specificity in knockout or knockdown systems?

A rigorous validation approach for DVL3 antibodies includes:

• Genetic knockout/knockdown controls:

  • Utilize DVL1/2/3-null HEK293 T-REx cells as negative controls to confirm antibody specificity

  • Implement siRNA or shRNA knockdown of DVL3 with quantitative assessment of signal reduction across applications

• Signal specificity assessment:

  • Confirm expected molecular weight (78 kDa calculated, typically observed at 88-93 kDa due to post-translational modifications)

  • Verify absence of bands in DVL3-depleted samples

  • Evaluate cross-reactivity with other Dishevelled family members, particularly important since some antibodies specifically do not cross-react with DVL2

• Functional validation approaches:

  • Correlate antibody signal with functional readouts such as TopFlash reporter assays measuring Wnt pathway activation

  • Assess whether the antibody detects expected changes in DVL3 complex formation following Wnt3a stimulation

• Recovery experiments:

  • Reintroduce tagged or untagged DVL3 into knockout systems and confirm signal restoration

  • Use multiple antibodies targeting different DVL3 epitopes to verify consistent detection patterns

• Publication record evaluation:

  • Consider published applications data, with some DVL3 antibodies having established records in WB (9 publications), IHC (2 publications), IF (2 publications), and IP (2 publications)

This comprehensive validation approach ensures experimental results can be interpreted with high confidence.

How can DVL3 antibodies be utilized to study Wnt signaling dynamics in live cells?

For studying real-time Wnt signaling dynamics using DVL3 antibodies:

• FlAsH-based sensor implementation:

  • Transfect cells with the DVL3 FlAsH III sensor construct

  • Label according to established protocols: incubate at 37°C for 1 hour with Hank's Balanced Salt Solution (HBSS) containing 500 nM FlAsH and 12.5 μM 1,2-ethanedithiol (EDT)

  • Reduce non-specific labeling with a 10-minute wash in HBSS containing 250 μM EDT

  • Complete with two HBSS washes before returning to DMEM medium

• Live-cell imaging protocol:

  • Establish baseline measurements before Wnt3a addition

  • Add Wnt3a and capture images at defined intervals (5, 15, 30, 60 minutes) to track complex formation kinetics

  • Incorporate appropriate controls (untreated, DKK1-treated) to distinguish specific Wnt-dependent changes

• Quantitative analysis approach:

  • Track DVL3 puncta formation and dynamics

  • Measure fluorescence correlation parameters to determine complex size changes

  • Analyze subcellular redistribution in response to pathway activation

• FRAP/FRET techniques integration:

  • Combine with FRAP (Fluorescence Recovery After Photobleaching) to assess DVL3 mobility changes

  • Implement FRET systems to detect interactions between DVL3 and other Wnt pathway components

This approach provides dynamic insights into DVL3's role in Wnt signaling that complement static biochemical analyses.

What roles do DVL3 expression patterns play in cancer pathology and how can they be analyzed?

DVL3 expression patterns in cancer contexts reveal important pathological insights:

• Expression level analysis methodology:

  • Implement immunohistochemistry with semiquantitative evaluation methods

  • Categorize expression as moderate (observed in 52.4% of glioblastomas) or strong (observed in 23.1% of cases)

  • Use immunoreactivity scoring systems for standardized assessment

• Subcellular localization patterns:

  • DVL3 predominantly localizes to the cytoplasm (97% of glioblastoma cases)

  • Nuclear co-expression occurs in 44% of samples

  • Strong expression significantly correlates with nuclear localization (P = 6.33 × 10^-5)

• Correlation with molecular profiles:

  • Analyze DVL3 expression in relation to other Wnt pathway components (e.g., sFRP3 as investigated in glioblastoma)

  • Compare expression patterns across different cancer subtypes and grades

• Tissue microarray applications:

  • Optimize antibody dilutions (1:500-1:2000) for high-throughput analysis

  • Implement automated scoring systems for reproducible quantification

• Prognostic significance assessment:

  • Correlate expression patterns with patient outcome data

  • Compare with established molecular markers for the specific cancer type

This multifaceted approach provides insights into DVL3's potential role in cancer pathogenesis and progression.

How do various post-translational modifications of DVL3 affect antibody binding and experimental outcomes?

Post-translational modifications (PTMs) of DVL3 significantly impact antibody recognition:

• Phosphorylation effects:

  • Extensive phosphorylation alters DVL3's apparent molecular weight from the calculated 78 kDa to observed 88-93 kDa in western blots

  • Kinase activity (particularly CK1ε) induces mobility shifts that may affect epitope accessibility

  • Use of phosphatase treatment as a control can help distinguish phosphorylation-dependent epitope masking

• Ubiquitination considerations:

  • Ubiquitination state affects DVL3 stability and complex formation

  • High molecular weight smears in western blots may indicate ubiquitinated forms

  • Deubiquitinating enzyme inhibitors may be necessary to preserve these modifications during sample preparation

• Protein conformation influence:

  • DVL3's participation in supermolecular complexes (0.4-2.0 MDa) may mask certain epitopes

  • Wnt3a stimulation induces conformational changes within 30 minutes that can alter antibody accessibility

• Antibody selection strategy:

  • For PTM-specific studies, select antibodies with epitopes distant from known modification sites

  • Consider using multiple antibodies targeting different regions to obtain comprehensive detection

• Experimental design adaptation:

  • Include appropriate controls (phosphatase treatment, kinase inhibitors like PF-670462)

  • Optimize sample preparation to preserve PTMs of interest

  • Consider native vs. denaturing conditions based on experimental questions

Understanding these PTM effects is crucial for accurate interpretation of experimental results, particularly in signaling studies where DVL3's modification state directly reflects pathway activity.

How can DVL3 antibodies be applied in studying cross-talk between Wnt signaling and other pathways?

For investigating signaling cross-talk involving DVL3:

• Co-immunoprecipitation strategy:

  • Use DVL3 antibodies for immunoprecipitation (0.5-4.0 μg for 1.0-3.0 mg of total protein lysate)

  • Probe for components of intersecting pathways (Notch, Hedgehog, Hippo, RTK)

  • Implement reverse co-IP validation to confirm interactions

• Stimulation experimental design:

  • Compare DVL3 complex composition under single vs. multiple pathway stimulation

  • Track temporal dynamics of complex assembly/disassembly following sequential pathway activation

  • Analyze how inhibition of one pathway affects DVL3's role in another

• Proximity ligation assay application:

  • Visualize endogenous protein-protein interactions in situ

  • Quantify interaction frequency in different subcellular compartments

  • Compare interaction patterns across normal vs. pathological conditions

• Combinatorial knockdown approach:

  • Utilize DVL1/2/3-null cell systems with reconstitution of specific variants

  • Assess how DVL3 manipulation affects signaling outputs from multiple pathways simultaneously

• Post-translational modification cross-regulation:

  • Investigate how kinases from different pathways affect DVL3 phosphorylation patterns

  • Analyze whether these modifications alter DVL3's scaffold function across pathways

This integrated approach reveals DVL3's role as a potential signaling hub coordinating multiple cellular processes.

What are the methodological considerations for studying DVL3 in primary patient samples?

When investigating DVL3 in primary patient samples:

• Tissue preservation protocol optimization:

  • For immunohistochemistry applications, optimize fixation timing to prevent epitope masking

  • Consider specialized preservation methods for phosphorylated DVL3 detection

• Antigen retrieval method selection:

  • Implement TE buffer pH 9.0 as the primary antigen retrieval method

  • Use citrate buffer pH 6.0 as an alternative approach when necessary

  • Validate retrieval efficiency for each tissue type

• Antibody dilution adaptation:

  • For IHC applications in patient samples, use 1:500-1:2000 dilution range

  • Titrate specifically for each tissue type to optimize signal-to-noise ratio

• Comparative analysis framework:

  • Include matched normal tissue controls when available

  • Establish standardized scoring systems (such as used in glioblastoma studies)

  • Correlate with other molecular markers and clinical parameters

• RNA-protein correlation assessment:

  • Perform parallel analysis of DVL3 mRNA expression (RT-qPCR, RNA-seq)

  • Compare protein localization with transcript abundance

  • Investigate potential post-transcriptional regulatory mechanisms

This methodological approach enables reliable analysis of DVL3's contribution to human pathologies while accounting for sample heterogeneity.

How do differences in DVL family members affect antibody selection for comparative studies?

For comparative studies of DVL family members:

• Specificity verification approach:

  • Select antibodies with documented specificity, such as those confirmed not to cross-react with other DVL family members

  • Validate specificity using overexpression and knockout controls for each DVL protein

  • Consider epitope mapping to understand potential cross-reactivity mechanisms

• Structural homology considerations:

  • DVL family members share conserved domains (DIX, PDZ, DEP) but differ in specific sequences

  • Target unique regions for differential detection

  • Account for isoform-specific variations that may affect antibody binding

• Expression level normalization:

  • Consider the relative abundance of different DVL proteins (DVL2 constitutes >95% of Dvl proteins in F9 cells)

  • Adjust antibody concentrations accordingly for fair comparison

  • Use recombinant protein standards for quantitative analysis

• Functional redundancy analysis:

  • Implement knockdown/knockout of individual DVL members to assess specific functions

  • Compare subcellular distributions of different DVL proteins under identical conditions

  • Analyze differential complex formation (DVL1 and DVL3 overexpression stimulates formation of large supermolecular complexes, while DVL2 shows different patterns)

• Systemic approach implementation:

  • Design experiments to simultaneously detect all DVL proteins in the same samples

  • Compare post-translational modification patterns across family members

  • Correlate with functional readouts to distinguish specific roles

This systematic comparison reveals both overlapping and distinct functions of DVL family members in Wnt signaling and beyond.