DVL3 Antibody, FITC conjugated

What is DVL3 and what is its fundamental role in cellular signaling?

DVL3 (Dishevelled 3) is a critical cytoplasmic phosphoprotein that functions as a key mediator in the Wnt signaling pathway. It plays an essential role in transducing signals from the frizzled receptors to downstream effectors, thereby influencing numerous cellular processes including cell proliferation, differentiation, and embryonic development. DVL3 is primarily localized in the cytoplasm where it facilitates signal transduction between membrane receptors and intracellular components . The protein contains several functional domains that enable its diverse interactions with other signaling molecules. Importantly, dysregulation of DVL3 and the Wnt pathway has been implicated in various pathological conditions, including cancer and neurodegenerative disorders, making it a significant target for research investigations .

What applications are most suitable for FITC-conjugated DVL3 antibodies?

FITC-conjugated DVL3 antibodies are particularly valuable for immunofluorescence microscopy, flow cytometry, and confocal imaging applications. The FITC (fluorescein isothiocyanate) conjugation provides direct fluorescent detection of DVL3 without requiring secondary antibodies, which simplifies experimental protocols and reduces background signal in multicolor imaging experiments . These conjugated antibodies are especially useful for studying DVL3 subcellular localization, trafficking dynamics, and protein-protein interactions in live or fixed cells. When investigating DVL3 conformational changes or translocation events in response to Wnt pathway activation, the FITC-conjugated antibody allows for real-time visualization of these processes. Additionally, these antibodies can be effectively employed in high-content screening assays to evaluate compounds that might modulate DVL3 function or localization .

How does DVL3 interact with other components of the Wnt signaling pathway?

DVL3 functions as a critical hub within the Wnt signaling network, integrating signals from upstream receptors and transmitting them to downstream effectors. Upon Wnt ligand binding to Frizzled receptors, DVL3 is recruited to the plasma membrane where it undergoes phosphorylation and conformational changes . These modifications alter DVL3's interaction capabilities with various binding partners. In the canonical Wnt pathway, activated DVL3 inhibits the β-catenin destruction complex (containing GSK3β), leading to β-catenin accumulation and subsequent transcriptional activation . Research has demonstrated that DVL3 conformational dynamics are heavily regulated by kinases, particularly Casein kinase 1ε (CK1ε), which phosphorylates DVL3 and induces structural changes from a "closed" to an "open" state . This conformational switching is crucial for proper signal transduction. Additionally, DVL3 interacts with components of non-canonical Wnt pathways, affecting cytoskeletal organization and cell polarity through distinct protein complexes .

What experimental validation has been performed on the DVL3 Antibody (4D3)?

The DVL3 Antibody (4D3) has undergone extensive validation to ensure specificity and reliability across multiple experimental applications. This mouse monoclonal IgG1 antibody was raised against amino acids 607-704 of Dvl-3 of mouse origin and has been confirmed to detect DVL3 in mouse, rat, and human samples . Validation studies have demonstrated its effectiveness in western blotting (WB) and immunoprecipitation (IP) applications, making it suitable for protein expression analysis and protein-protein interaction studies . The antibody's specificity has been verified through the detection of the expected ~85-90 kDa band corresponding to DVL3 protein in western blots across multiple cell types. Cross-reactivity testing with related Dishevelled family members (DVL1 and DVL2) has confirmed its selectivity for DVL3. The FITC-conjugated version retains the specificity of the parent antibody while providing direct fluorescent detection capabilities .

How can I analyze DVL3 conformation dynamics in living cells?

Analyzing DVL3 conformation dynamics in living cells requires sophisticated techniques that can capture real-time molecular changes. A particularly effective approach involves FRET (Förster Resonance Energy Transfer)-based biosensors specifically designed for DVL3. As demonstrated in recent research, FlAsH-based FRET represents a powerful technique for monitoring protein conformation in biological setups where large tags might interfere with protein complex formation and function . To implement this approach, you would need to generate DVL3 sensors containing an ECFP (Enhanced Cyan Fluorescent Protein) tag at the N-terminus and a CCPGCC tag inserted at strategic locations within the protein's structure, especially within intrinsically disordered regions .

The FlAsH molecule forms a fluorescent complex with the CCPGCC sequence that can be monitored in real-time and controlled with BAL (British Anti-Lewisite) addition to shut off the signal. In experimental settings, researchers have successfully designed four different DVL3 sensors (FlAsH I-IV) with varying CCPGCC tag positions . These sensors showed minimal interference with wild-type DVL3 biological properties, including activation of the Wnt/β-catenin pathway, CK1ɛ-dependent electrophoretic mobility, and changes in subcellular localization. The FlAsH III sensor demonstrated particularly robust responses to conformational changes induced by various stimuli, making it ideal for investigating DVL3 dynamics .

For your experiments, ensure appropriate controls for distinguishing between intramolecular and intermolecular FRET, as DVL3 can undergo multimerization via its DIX and DEP domains.

How does Casein kinase 1ε (CK1ε) regulate DVL3 conformation and function?

CK1ε plays a dual critical role in regulating DVL3 conformation and function through both physical interaction and phosphorylation-mediated mechanisms. Research utilizing FRET-based DVL3 sensors has revealed that CK1ε can maintain DVL3 in distinct conformational states depending on its activity status . When CK1ε is present but inhibited (either pharmacologically with PF670462 or by using dominant negative mutants), DVL3 adopts a more compact "closed" conformation with increased FRET efficiency. Conversely, active CK1ε induces phosphorylation of DVL3, triggering a transition to a more "open" conformation characterized by decreased FRET efficiency .

Intriguingly, complete absence of CK1ε (as demonstrated in CK1ɛ knockout cells) results in DVL3 remaining in an open conformation despite being non-phosphorylated, which differs from the closed conformation observed when CK1ε is present but inhibited . This suggests that CK1ɛ has two distinct functions: (1) retaining DVL3 in a compact conformation through physical interaction when inactive, and (2) triggering a phosphorylation-induced open conformation when activated .

For experimental investigation of this regulatory mechanism, researchers should consider using both pharmacological inhibitors like PF670462 (10 μM) and genetic approaches such as dominant negative CK1ε mutants (particularly the P3 mutant) or CRISPR-Cas9-generated CK1ε knockout cell lines. Monitoring DVL3 conformational changes in response to these manipulations can be achieved using the FlAsH III FRET sensor, which has shown the most prominent differences in FRET efficiency under varying CK1ε conditions .

What methodologies are effective for studying DVL3 in inflammatory responses?

Investigating DVL3's role in inflammatory responses requires a multifaceted approach combining in vitro and in vivo methodologies. Recent research has revealed that DVL3, in conjunction with Wnt3a signaling, plays an unexpected anti-inflammatory role in TLR4-mediated immune responses . To study this effectively, consider the following integrated methodological approach:

For cellular models, primary monocytes or macrophage cell lines treated with LPS to activate TLR4 signaling provide an excellent system to investigate DVL3 function in inflammation. Inhibition or silencing of DVL3 can be achieved through specific siRNA or chemical inhibitors of the Wnt pathway (such as IWP-2 and PNU-74654) . Key readouts should include:

• Pro-inflammatory cytokine production (IL-12, IL-6, TNFα) measured by ELISA or multiplexed assays

• Analysis of β-catenin accumulation using western blotting

• Assessment of NF-κB P65 phosphorylation and DNA binding activity through phospho-specific antibodies and EMSA/ChIP assays

• Evaluation of downstream signaling molecules including GSK3β

For in vivo validation, murine endotoxin models provide relevant systems to confirm cellular findings. The inflammatory response can be measured through:

• Systemic pro-inflammatory cytokine levels in serum

• Neutrophil infiltration in tissues using immunohistochemistry with FITC-conjugated antibodies against neutrophil markers like Ly6G

• Tissue damage assessment in organs commonly affected by inflammation

Gain- and loss-of-function approaches are particularly valuable, including ectopic expression of DVL3, GSK3β, and β-catenin to establish causality in the observed phenotypes. The combination of these methodologies provides comprehensive insights into how DVL3 functions as a rheostat to restrain the activity of NF-κB during inflammatory responses .

How can the C-terminus-PDZ interaction of DVL3 be experimentally analyzed?

The interaction between DVL3's C-terminus and its PDZ domain represents a critical mechanism controlling DVL3 conformational states, with significant implications for Wnt signaling regulation . To experimentally analyze this interaction, researchers should consider these methodological approaches:

First, generating truncation or deletion mutants of DVL3 lacking the conserved terminal sequence (equivalent to the EFFVDIM sequence in mouse Dvl1) allows for direct assessment of how this region contributes to protein conformation and function . These mutants can be analyzed for changes in protein folding, interaction capabilities, and signaling activity.

For protein-protein interaction analysis, in vitro approaches such as GST-pulldown assays using purified PDZ domains and C-terminal peptides can confirm direct binding. This can be complemented with isothermal titration calorimetry (ITC) or surface plasmon resonance (SPR) to determine binding affinities and kinetics.

In cellular contexts, proximity ligation assays (PLA) offer a sensitive method to visualize and quantify the C-terminus-PDZ interaction in situ. Additionally, introducing competing peptides mimicking the C-terminal sequence can disrupt this interaction and provide insights into its functional significance .

Finally, structural studies using X-ray crystallography or NMR spectroscopy of the PDZ domain bound to C-terminal peptides can provide atomic-level details of this interaction, which may guide the design of specific modulators for research applications.

What technical considerations are important when using DVL3 antibodies in FRET-based assays?

When employing DVL3 antibodies in FRET-based assays, several critical technical considerations must be addressed to ensure reliable and interpretable results. First, the choice of FRET pair is paramount—the FITC-conjugated DVL3 antibody works effectively as a donor with appropriate acceptor fluorophores having sufficient spectral overlap, such as tetramethylrhodamine (TRITC) or Cy3 .

Antibody concentration requires careful optimization, as too high concentrations can lead to non-specific binding and increased background, while too low concentrations may yield insufficient signal. Titration experiments are essential to determine optimal concentrations, typically ranging from 1-5 μg/ml for most applications .

Controls for specific binding are crucial—these should include isotype controls, blocking peptide competition assays, and validation in knockout/knockdown cells to confirm specificity. Additionally, positive controls using cells known to express high levels of DVL3 should be included .

Potential interference from DVL3 multimerization must be addressed, as DVL3 can form oligomers via its DIX and DEP domains, potentially leading to intermolecular FRET that could confound intramolecular FRET measurements. This can be controlled for by comparing FRET signals between full FRET-sensor constructs and co-expressed separate donor and acceptor components .

For fixed cell applications, fixation methods significantly impact FRET efficiency—paraformaldehyde (4%) generally preserves fluorophore activity well, while harsher fixatives may denature fluorescent proteins or affect antibody binding sites .

In live cell applications, photobleaching concerns must be mitigated through minimizing exposure times, using anti-fade reagents, and implementing proper experimental design to account for potential signal decay over time .

The table below summarizes key parameters for optimization when using DVL3 antibody, FITC conjugated in FRET assays:

How can I troubleshoot inconsistent results when using DVL3 antibodies for studying Wnt pathway activation?

Inconsistent results when using DVL3 antibodies to study Wnt pathway activation can stem from multiple factors that require systematic troubleshooting. One primary consideration is antibody batch variability, which can significantly impact experimental outcomes. Different lots may have varying affinities or epitope recognition properties. To address this, maintain consistent antibody lots throughout a study series and validate each new lot against previous standards using positive control samples .

Cell-specific variations in DVL3 expression and post-translational modifications can also lead to inconsistencies. Different cell lines may express various levels of DVL3 or possess distinct patterns of DVL3 modification. Establish baseline expression profiles for your specific cell models using quantitative western blotting before proceeding with functional studies .

Wnt stimulation protocols represent another source of variability, as different Wnt ligands activate distinct downstream pathways with varying kinetics. For consistent results, standardize the source, concentration, and treatment duration of Wnt ligands. Commercial recombinant Wnt proteins may lose activity over time, so proper storage and regular validation of activity are essential .

Inadequate controls often contribute to inconsistent findings. Always include positive controls (cells with known DVL3 activation), negative controls (untreated cells), and when possible, DVL3 knockout/knockdown controls to establish specificity of the observed signals .

Technical parameters during immunodetection procedures should be carefully controlled, including fixation methods, permeabilization conditions, blocking reagents, antibody concentrations, and incubation times. Document and standardize these parameters across experiments .

For phosphorylation-dependent events, ensure that phosphatase inhibitors are consistently included in lysis buffers to prevent post-lysis dephosphorylation. The timing between cell stimulation and lysis is critical, as DVL3 phosphorylation states are dynamic and time-dependent .

What controls should be included when studying DVL3 phosphorylation and its impact on protein conformation?

When investigating DVL3 phosphorylation and its effects on protein conformation, implementing comprehensive controls is essential for generating reliable and interpretable data. First, kinase activity controls should be established to validate the specific contribution of kinases like CK1ε. This involves parallel experiments with kinase inhibitors (such as PF670462 for CK1δ/ε), kinase-dead mutants (dominant negative constructs like CK1ε-P3), and CK1ε knockout cell lines .

Phosphorylation site controls are crucial for mechanistic understanding. These include phospho-deficient mutants (with Ser/Thr residues mutated to Ala) and phospho-mimetic mutants (with Ser/Thr residues mutated to Asp/Glu) at key regulatory sites. Comparing these mutants allows for dissection of specific phosphorylation events' contributions to conformational changes .

For FRET-based conformational studies, proper FRET controls are indispensable. These include: donor-only and acceptor-only samples to establish baseline fluorescence; intermolecular FRET controls using separately expressed fluorophores to distinguish between intra- and intermolecular FRET; and BAL treatment controls for FlAsH-based systems to confirm specific FlAsH-tetracysteine binding .

Cell type controls help determine the generalizability of findings across biological systems. Testing DVL3 phosphorylation and conformation in multiple cell types with varying levels of endogenous kinases and phosphatases provides crucial context for interpretations .

Temporal controls are necessary as phosphorylation is a dynamic process. Time-course experiments following stimulation or inhibition allow tracking of the kinetic relationship between phosphorylation events and conformational changes .

The following table summarizes essential controls for studying DVL3 phosphorylation and conformation:

How might DVL3 antibodies be utilized in developing therapeutic approaches targeting the Wnt pathway?

DVL3 antibodies, particularly their FITC-conjugated variants, hold significant potential for developing targeted therapeutic approaches against Wnt pathway dysregulation in various diseases. As a central hub in Wnt signaling, DVL3 represents a strategic intervention point that may offer advantages over targeting upstream or downstream components . Future therapeutic development could leverage DVL3 antibodies in several innovative ways.

For cancer therapeutics, DVL3 antibodies may enable precise targeting of aberrant Wnt activation, which drives tumor growth in multiple malignancies. By developing antibody-drug conjugates (ADCs) using anti-DVL3 antibodies, researchers could potentially deliver cytotoxic payloads specifically to cells with dysregulated DVL3 expression or activation . The FITC conjugation technology could be adapted to incorporate near-infrared fluorophores for image-guided surgical interventions, helping surgeons visualize tumor margins expressing elevated DVL3 levels.

In inflammatory disorders, the newly discovered anti-inflammatory function of Wnt3a-DVL3 signaling suggests potential for DVL3-targeted immunomodulatory therapies . Antibodies that selectively enhance DVL3's restraining effect on NF-κB activation could help dampen excessive inflammatory responses in conditions like sepsis or autoimmune diseases. This approach would require antibodies that stabilize specific DVL3 conformations associated with anti-inflammatory activity.

For neurodegenerative diseases, where Wnt pathway impairment has been implicated, conformation-specific DVL3 antibodies might help restore proper signaling. These could be designed to recognize and stabilize the active conformation of DVL3, potentially compensating for upstream defects in the pathway .

The development of intrabodies (intracellular antibodies) derived from DVL3 antibodies represents another frontier. These engineered antibody fragments could be delivered using cell-penetrating peptides or gene therapy approaches to modulate DVL3 conformation and function within cells, potentially correcting signaling abnormalities with greater specificity than small molecule inhibitors .

What novel experimental approaches could enhance our understanding of DVL3 conformational dynamics?

Advancing our understanding of DVL3 conformational dynamics requires innovative experimental approaches that build upon existing knowledge while introducing new technological capabilities. Single-molecule FRET (smFRET) represents a transformative approach that could reveal previously undetectable conformational states by eliminating population averaging inherent in bulk measurements . This technique would allow researchers to track individual DVL3 molecules in real-time, potentially revealing rare or transient conformational intermediates critical for signaling.

Cryo-electron microscopy (cryo-EM) applied to DVL3 complexes could provide unprecedented structural insights, particularly for assemblies too large or flexible for crystallography. Recent advances in cryo-EM resolution now enable visualization of dynamic protein regions, potentially allowing researchers to capture DVL3 in different conformational states when bound to various interaction partners .

Integrating hydrogen-deuterium exchange mass spectrometry (HDX-MS) with computational modeling would offer complementary structural information. HDX-MS can map solvent-accessible regions of DVL3 under different conditions (with/without kinase activity, Wnt stimulation, etc.), while molecular dynamics simulations could predict conformational transitions and energy landscapes .

Optogenetic approaches to control DVL3 conformation represent another frontier. By incorporating light-sensitive domains into strategic positions within DVL3, researchers could trigger conformational changes with precise spatial and temporal control, allowing for cause-effect studies of specific conformational states on downstream signaling events .

Proximity labeling techniques like BioID or APEX2 fused to DVL3 could map the conformation-specific interactome of DVL3. These approaches would identify proteins that interact with DVL3 only in specific conformational states, providing insights into how conformational changes alter signaling complex assembly .

Lastly, developing nanobodies (single-domain antibodies) that recognize specific DVL3 conformations could provide powerful tools for both detection and manipulation of DVL3 states. Such conformation-specific binders could be used diagnostically to track DVL3 activation states or therapeutically to stabilize particular conformations associated with desired signaling outcomes .