DVL2 Antibody

What are the validated applications for DVL2 antibodies in laboratory research?

DVL2 antibodies have been validated for multiple experimental applications including Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence/Immunocytochemistry (IF/ICC), Immunoprecipitation (IP), Co-Immunoprecipitation (CoIP), and ELISA assays. The validated dilution ranges for these applications are:

When designing experiments, it is essential to optimize antibody concentration for your specific cell line or tissue type as reactivity may vary between samples .

What species reactivity has been confirmed for DVL2 antibodies?

DVL2 antibodies have been tested and confirmed to have reactivity with human, mouse, and rat samples. Published literature also cites reactivity with pig and xenopus models, expanding potential research applications across multiple species. This cross-species reactivity makes DVL2 antibodies valuable tools for comparative studies between different model organisms .

How should DVL2 antibodies be stored and handled to maintain optimal activity?

For optimal preservation of antibody activity, DVL2 antibodies should be stored at -20°C in a buffer containing PBS with 0.02% sodium azide and 50% glycerol at pH 7.3. Under these conditions, the antibody remains stable for one year. Repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and reduced antibody performance. Working aliquots may be prepared for frequent use to prevent degradation of the primary stock .

What controls should be included when using DVL2 antibodies for knockdown/knockout validation?

When using DVL2 antibodies in knockdown or knockout studies, positive and negative controls are essential for result validation. Multiple publications have used DVL2 antibodies for KD/KO verification. Effective controls include:

• Positive control: Lysates from cells known to express DVL2 (e.g., HEK-293, MCF-7, or HepG2 cells)

• Negative control: Lysates from the same cell line with DVL2 knockdown using siRNA or CRISPR-Cas9

• Loading control: Probing for a housekeeping protein (e.g., GAPDH, β-actin) to ensure equal loading

The difference in band intensity between control and KD/KO samples provides quantitative validation of both the knockdown efficiency and antibody specificity .

What antigen retrieval methods are recommended for DVL2 immunohistochemistry?

For optimal DVL2 detection in tissue sections, antigen retrieval is a critical step. The recommended protocol involves using TE buffer at pH 9.0. Alternatively, citrate buffer at pH 6.0 may be used, though results may vary depending on tissue fixation methods and sample age. Antigen retrieval optimization may be necessary for specific tissue types, particularly for formalin-fixed paraffin-embedded (FFPE) samples where protein epitopes may be masked by crosslinking .

How does DVL2 expression correlate with immune markers in HER2-positive breast cancer?

Recent studies have revealed significant correlations between DVL2 expression and immune markers in HER2-positive breast cancer. Analysis of clinical samples demonstrated that higher DVL2 expression at baseline biopsy showed a significant negative correlation with CD8α levels (r = -0.67, p < 0.05), indicating potential immunosuppressive effects. Conversely, DVL2 expression positively correlated with neutrophil-to-lymphocyte ratio (NLR) (r = 0.58, p < 0.05), a clinical parameter where higher values indicate worse cancer prognosis. These findings suggest DVL2 may play a role in modulating the tumor immune microenvironment, potentially contributing to immune evasion in breast cancer .

What experimental approaches can be used to study DVL2's role in cancer cell proliferation?

To investigate DVL2's impact on cancer cell proliferation, researchers can employ multiple complementary approaches:

• Loss-of-function studies using siRNA or shRNA targeting DVL2

• Live cell imaging to monitor proliferation rates over time

• Cell cycle analysis using flow cytometry to quantify cell distribution across G1, S, and G2/M phases

• RT-qPCR analysis of Wnt target genes involved in proliferation

• Western blot analysis of downstream signaling proteins

• Combinatorial studies with targeted therapies (e.g., Neratinib for HER2+ breast cancer)

This multi-faceted approach has revealed that DVL2 knockdown results in reduced proliferation, higher growth arrest in G1 phase, and limited mitosis (G2/M), supporting DVL2's pro-proliferative role in cancer cells .

What tissue types have been validated for DVL2 antibody immunohistochemistry in cancer research?

DVL2 antibodies have been validated for immunohistochemistry in multiple cancer tissue types, including:

• Human lung cancer tissue

• Human gliomas tissue

• Human prostate cancer tissue

• Human breast cancer tissue (particularly HER2-positive samples)

Additionally, normal mouse and rat colon tissues have been validated for comparative studies. When working with these tissue types, researchers should follow the recommended antigen retrieval methods (TE buffer pH 9.0 or citrate buffer pH 6.0) and antibody dilutions (1:50-1:500) for optimal results .

How does DVL2 modulate inflammatory cytokine production and what methodologies best capture this effect?

DVL2 functions as a negative regulator of inflammatory cytokine production, particularly in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). To effectively study this modulatory effect, researchers should employ multiple complementary methods:

• ELISA analysis of cell supernatants to quantify secreted cytokines (IL-1β, IL-6, IL-8)

• RT-PCR for measuring cytokine mRNA levels

• RNA-seq to comprehensively analyze altered gene expression patterns

• Stimulation with TNF-α to activate the NF-κB pathway and observe DVL2's inhibitory effects

Research has demonstrated that DVL2 overexpression reduces the release of IL-6 both with and without TNF-α stimulation, while IL-1β and IL-8 inhibition becomes apparent only upon TNF-α stimulation. This suggests DVL2's anti-inflammatory effects are most pronounced in inflammatory contexts .

What molecular mechanisms underlie DVL2's regulation of the NF-κB pathway in inflammatory conditions?

DVL2 inhibits the NF-κB pathway through multiple molecular mechanisms that can be investigated through the following experimental approaches:

• FlowSight analysis to assess nuclear translocation of P65

• Immunoprecipitation (IP) to detect direct interaction between DVL2 and P65

• Chromatin immunoprecipitation (ChIP) to evaluate P65 binding to promoters of target genes

• RNA-seq to identify altered expression of NF-κB pathway genes

Research findings demonstrate that DVL2 overexpression inhibits TNF-α-induced nuclear translocation of P65, a crucial step in NF-κB pathway activation. Furthermore, IP analysis confirms direct interaction between DVL2 and P65 in RA-FLSs. ChIP assays reveal that DVL2 inhibits TNF-α-induced binding of P65 to promoters of anti-apoptotic and inflammatory genes, providing a mechanistic explanation for DVL2's anti-inflammatory effects .

How can researchers investigate the dual role of DVL2 in apoptosis and inflammation?

To effectively study DVL2's dual role in regulating both apoptosis and inflammation, researchers should implement a comprehensive experimental approach:

• Apoptosis analysis:

  • Flow cytometry with Annexin V/PI staining

  • Western blot for apoptotic markers (cleaved caspase-3, PARP)

  • RT-PCR for anti-apoptotic genes (A20, GADD45β, cIAP1, cIAP2)

• Inflammation analysis:

  • ELISA for secreted cytokines

  • RT-PCR for inflammatory cytokine expression

  • NF-κB pathway activation assessment

  • RNA-seq for global gene expression changes

Studies have revealed that DVL2 overexpression decreases mRNA levels of NF-κB-dependent anti-apoptotic genes (A20, GADD45β, cIAP2) and inflammatory cytokines (IL-1β, IL-6, IL-8), particularly upon TNF-α stimulation. This indicates that DVL2 promotes apoptosis while simultaneously inhibiting inflammatory responses, possibly through its inhibitory effect on the NF-κB pathway .

How should researchers address potential cross-reactivity between DVL paralogs when using DVL2 antibodies?

When working with DVL2 antibodies, researchers must consider potential cross-reactivity with other DVL paralogs (DVL1 and DVL3) due to sequence homology. To address this challenge:

• Validate antibody specificity using knockout/knockdown controls for each DVL paralog

• Perform Western blot analysis to verify that the antibody detects a band at the expected molecular weight for DVL2 (90-95 kDa) rather than DVL1 or DVL3

• Consider using multiple antibodies targeting different epitopes of DVL2

• Include recombinant DVL1, DVL2, and DVL3 proteins as controls to assess cross-reactivity

In functional studies, consider the redundancy between DVL paralogs by evaluating the expression levels of all three proteins and potentially employing combinatorial knockdowns to fully understand their collective contributions to the observed phenotypes .

What methodological approaches can resolve discrepancies between calculated and observed molecular weights of DVL2?

The discrepancy between the calculated molecular weight of DVL2 (79 kDa) and the observed weight in experimental conditions (90-95 kDa) requires careful methodological consideration:

• Analyze post-translational modifications:

  • Phosphorylation status using phospho-specific antibodies or phosphatase treatment

  • Ubiquitination profile using ubiquitin-specific antibodies or deubiquitinase treatment

  • Glycosylation analysis using glycosidase treatments

• Optimize SDS-PAGE conditions:

  • Test different acrylamide percentages (8-10% for proteins >70 kDa)

  • Adjust running buffer composition and pH

  • Modify sample preparation (denaturing conditions, reducing agents)

• Include recombinant DVL2 protein as a reference standard

This comprehensive approach can help researchers accurately identify DVL2 bands and understand the molecular basis for the observed weight discrepancy .

How can researchers optimize DVL2 antibody performance across different experimental platforms?

To achieve optimal DVL2 antibody performance across multiple experimental platforms, researchers should implement systematic optimization strategies:

Each experimental system may require specific optimization to obtain optimal results. It is recommended that researchers perform preliminary validation experiments to determine the ideal conditions for their specific sample types and experimental questions .

How does current research on DVL2 inform potential therapeutic targeting in cancer and inflammatory diseases?

Current research suggests DVL2 as a promising therapeutic target with dual potential in cancer and inflammatory diseases. In HER2-positive breast cancer, DVL2 appears to promote cancer cell proliferation and modulate the tumor immune microenvironment, with higher expression correlating with decreased CD8+ T cell infiltration and worse prognostic markers. Conversely, in inflammatory conditions like rheumatoid arthritis, DVL2 overexpression inhibits inflammatory cytokine production and promotes apoptosis through NF-κB pathway inhibition.

This dual role suggests context-dependent therapeutic approaches:

• In cancer: DVL2 inhibition might reduce cancer cell proliferation and enhance anti-tumor immunity

• In inflammatory diseases: DVL2 activation could reduce inflammatory cytokine production and promote resolution of inflammation

Future therapeutic development will require further understanding of tissue-specific functions and pathway crosstalk to optimize targeting strategies .

What emerging methodologies might enhance DVL2 research beyond current antibody-based approaches?

While antibody-based approaches remain fundamental to DVL2 research, emerging methodologies offer promising avenues for deeper mechanistic insights:

• CRISPR-Cas9 gene editing for precise DVL2 knockout or mutation

• Proximity labeling techniques (BioID, APEX) to identify novel DVL2 interacting partners

• Single-cell RNA-seq to characterize DVL2's cell type-specific functions

• Super-resolution microscopy for detailed subcellular localization

• Patient-derived organoids to study DVL2 in physiologically relevant models

• Phospho-proteomics to map DVL2 signaling networks

• In vivo imaging with reporter systems to monitor DVL2 activity in real-time

These advanced approaches can complement traditional antibody-based methods to provide more comprehensive understanding of DVL2's complex biological functions across different cellular contexts and disease states .

How can researchers better integrate DVL2 studies with broader Wnt pathway research?

To effectively integrate DVL2 research within the broader context of Wnt signaling, researchers should adopt multifaceted approaches that connect DVL2-specific findings to established Wnt pathway mechanisms:

• Investigate DVL2's interactions with canonical and non-canonical Wnt pathway components:

  • β-catenin stabilization and nuclear translocation

  • JNK pathway activation

  • Calcium signaling modulation

• Compare and contrast DVL paralogs (DVL1, DVL2, DVL3):

  • Paralog-specific functions

  • Redundancy and compensation mechanisms

  • Tissue-specific expression patterns

• Explore crosstalk between Wnt/DVL2 and other signaling pathways:

  • NF-κB pathway (established in inflammatory contexts)

  • HER2 signaling (relevant in breast cancer)

  • TNF signaling (for inflammation regulation)

• Develop integrated multi-omics approaches:

  • Combine transcriptomics, proteomics, and epigenomics

  • Correlate DVL2 expression with global Wnt pathway activity

  • Map DVL2-dependent signaling networks