WWC3 Antibody

What is WWC3 and why is it important in cancer research?

WWC3 (WW and C2 domain containing 3) serves as a tumor suppressor protein that plays a significant role in activating the Hippo signaling pathway. Research has shown that WWC3 downregulation correlates with poor prognosis in gastric cancer and potentially other malignancies. The protein inhibits cell proliferation and invasion capabilities while regulating cell cycle progression, making it a valuable target for cancer research . Experimental evidence demonstrates that WWC3 represses cyclin D1 and cyclin E expression while upregulating p27, suggesting its importance in cell cycle control mechanisms.

What are the optimal methods for WWC3 protein detection in tissue samples?

The primary methods for WWC3 protein detection in tissue samples include immunohistochemistry (IHC) and Western blot (WB). For IHC applications, researchers typically employ a standardized protocol using WWC3 rabbit polyclonal antibody at 1:200 dilution (such as those from Sigma-Aldrich), followed by polymer secondary antibodies and DAB development. Counterstaining with hematoxylin provides cellular context. For optimal results, tissue samples should undergo proper fixation, antigen retrieval with citrate buffer, and endogenous peroxidase blocking with H₂O₂ . Cytoplasmic immunopositivity is considered positive WWC3 staining, with scoring systems based on both intensity and percentage of positive cells.

How should WWC3 antibody specificity be validated in experimental settings?

Validation of WWC3 antibody specificity requires a multi-pronged approach. Researchers should:

• Compare staining patterns between tumor tissues and corresponding normal tissues

• Perform parallel Western blots with the same antibody used for IHC

• Include positive and negative controls in experimental designs

• Test the antibody in cells with WWC3 overexpression and in those with WWC3 depletion via siRNA

This validation approach confirms that the antibody demonstrates increased signal intensity with overexpression and decreased signal with depletion, producing single bands at the expected molecular weight when analyzed by Western blot . Antibody cross-reactivity with related proteins (WWC1, WWC2) should also be evaluated.

What is the recommended scoring system for WWC3 immunohistochemical analysis in tumor samples?

For advanced quantitative assessment of WWC3 expression by IHC, a semi-quantitative scoring system is recommended. This system evaluates both staining intensity and percentage of positive tumor cells:

The final score is calculated by multiplying the intensity score by the percentage score, yielding values from 0-12. Tumors are then classified as having low expression (final score <6) or high expression (final score ≥6) . This standardized approach enables consistent assessment across different studies and facilitates statistical analysis of WWC3 expression in relation to clinicopathological parameters.

How can WWC3 antibody be effectively used to study Hippo signaling pathway activation?

To effectively study WWC3's role in Hippo signaling pathway activation, researchers should employ a multi-method approach:

• Combine WWC3 antibody-based protein detection with functional assays measuring TEAD transcriptional activity

• Assess nuclear versus cytoplasmic localization of YAP protein following WWC3 manipulation

• Quantify expression levels of downstream targets like CTGF using Western blot

• Employ luciferase reporter assays to measure TEAD-dependent transcription

Research shows that WWC3 activates Hippo signaling by suppressing TEAD transcription activity, resulting in downregulation of both total and nuclear YAP and its target CTGF . When designing experiments to study this pathway, researchers should include appropriate controls and consider the temporal dynamics of signaling events following WWC3 manipulation.

What are the critical considerations when using WWC3 antibody in studies involving cell cycle regulation?

When using WWC3 antibody to investigate cell cycle regulation, researchers must consider several critical factors:

• Cell synchronization: Synchronize cells at specific cell cycle phases to accurately assess WWC3's impact

• Multi-parameter analysis: Combine antibody-based WWC3 detection with flow cytometry for cell cycle analysis

• Temporal dynamics: Establish optimal timepoints for analysis following WWC3 manipulation

• Downstream effector monitoring: Simultaneously assess cyclin D1, cyclin E, and p27 expression levels

Research has demonstrated that WWC3 overexpression inhibits cell cycle progression at the G1/S transition, while its depletion accelerates this transition . When designing experiments, include appropriate controls and consider potential cell type-specific differences in WWC3 function.

What are the optimal transfection conditions for WWC3 overexpression and depletion studies?

For WWC3 overexpression studies, the following protocol has proven effective:

• Utilize WWC3 plasmid (such as those available from OriGene)

• Transfect using Lipofectamine 3000 reagent following manufacturer's protocols

• Include empty vector (pCMV6) as negative control

• Harvest cells 48-72 hours post-transfection for optimal expression

For WWC3 depletion studies:

• Use siGENOME siRNA pool for WWC3 (such as M-013869-01-0005 from Dharmacon)

• Transfect using DharmaFECT 1 reagent according to manufacturer's protocol

• Include non-targeting siRNA as negative control

• Harvest cells 48 hours post-transfection for analysis

Verification of WWC3 manipulation should be performed via both qRT-PCR and Western blot to confirm successful overexpression or knockdown.

How can researchers resolve inconsistent WWC3 antibody staining patterns in tissue samples?

Inconsistent WWC3 antibody staining can result from several factors. To resolve these issues:

• Optimize antigen retrieval methods: Test different buffers (citrate vs. EDTA) and durations

• Adjust antibody concentration: Perform titration experiments to determine optimal dilution

• Modify incubation conditions: Test different temperatures and durations for primary antibody incubation

• Enhance blocking steps: Use more effective blocking reagents to reduce background staining

• Standardize tissue processing: Ensure consistent fixation times and processing methods

Additionally, batch testing antibodies from different lots and including positive and negative controls in each staining run can help identify sources of variability . For challenging samples, signal amplification methods may be necessary to detect low-level WWC3 expression.

What methodological approaches can resolve conflicting data regarding WWC3 expression and function?

When facing conflicting data about WWC3 expression or function, researchers should implement these methodological approaches:

• Employ multiple antibodies targeting different WWC3 epitopes to confirm expression patterns

• Use complementary detection methods (IHC, WB, IF, qRT-PCR) to validate findings

• Test WWC3 function across multiple cell lines to identify cell type-specific effects

• Implement rescue experiments by reintroducing WWC3 after knockdown to confirm specificity

• Design domain-specific mutations to map functional regions responsible for observed effects

Research has shown potential variations in WWC3 function across different cancer types and cellular contexts . When publishing findings, researchers should clearly report antibody specifications, experimental conditions, and potential limitations to facilitate interpretation of seemingly contradictory results.

How can WWC3 antibody be utilized to study correlations between protein expression and patient prognosis?

To effectively study correlations between WWC3 expression and patient outcomes:

• Employ tissue microarrays with adequate sample size and follow-up data

• Implement standardized scoring systems as detailed in section 2.1

• Conduct Kaplan-Meier survival analysis comparing high versus low WWC3 expression groups

• Perform multivariate Cox regression to determine if WWC3 is an independent prognostic factor

Research has demonstrated that WWC3 downregulation correlates with advanced stage, positive nodal status, higher relapse rate, and poor survival in gastric cancer patients . When designing such studies, ensure appropriate statistical power and account for potential confounding variables such as treatment differences.

What are the recommended protocols for studying WWC3's impact on cell invasion and migration?

To study WWC3's effects on cell invasion and migration:

• Transwell invasion assays: Use Matrigel-coated chambers to assess invasive capacity

• Wound healing assays: Measure migration rate after creating a standardized "wound"

• 3D spheroid invasion assays: Evaluate invasion in more physiologically relevant models

• Live-cell imaging: Monitor real-time changes in cell movement and morphology

When conducting these assays, cells should be manipulated to either overexpress or deplete WWC3, with appropriate vector or siRNA controls . Quantification should be performed using standardized image analysis protocols, with multiple biological and technical replicates to ensure reproducibility.

How can researchers effectively investigate cross-talk between WWC3 and other Hippo pathway components?

To investigate cross-talk between WWC3 and other Hippo pathway components:

• Co-immunoprecipitation: Identify direct protein-protein interactions using WWC3 antibody

• Proximity ligation assays: Visualize protein interactions in situ

• Sequential knockdown/overexpression: Manipulate multiple pathway components to identify epistatic relationships

• Phosphorylation-specific antibodies: Monitor activation states of key pathway components

Research has shown that WWC3 regulates YAP localization and TEAD transcriptional activity . When designing experiments to study pathway cross-talk, researchers should consider the temporal dynamics of signaling events and potential compensatory mechanisms involving related proteins such as WWC1 and WWC2.

How can WWC3 antibody applications be integrated with mass spectrometry-based proteomics?

Integration of WWC3 antibody applications with mass spectrometry-based proteomics can be achieved through:

• Immunoprecipitation-mass spectrometry (IP-MS): Use WWC3 antibody to pull down protein complexes for MS analysis

• Reverse phase protein arrays (RPPA): Quantify WWC3 and related proteins across multiple samples simultaneously

• Proximity-dependent biotin identification (BioID): Identify proteins in close proximity to WWC3

• Phosphoproteomics: Identify phosphorylation changes in the Hippo pathway following WWC3 manipulation

These approaches provide complementary data to traditional antibody-based methods and can reveal novel interaction partners and signaling mechanisms that may not be detected by targeted approaches alone.

What considerations are important when using WWC3 antibody in multiplexed imaging studies?

For multiplexed imaging studies using WWC3 antibody:

• Antibody compatibility: Ensure WWC3 antibody is compatible with multiplexing technology (e.g., spectral unmixing, sequential staining)

• Cross-reactivity assessment: Test for cross-reactivity with other antibodies in the multiplexed panel

• Signal amplification: Consider signal amplification methods for detecting low-abundance WWC3

• Optimization of antigen retrieval: Determine if single or multiple antigen retrieval methods are needed

• Validation of multiplexed signals: Confirm that multiplexed detection matches single-plex controls

Multiplexed imaging enables simultaneous visualization of WWC3 with other Hippo pathway components, cell cycle markers, or tumor microenvironment elements, providing spatial context to functional relationships that cannot be achieved with bulk analysis methods.