WDR66 Antibody

What is WDR66 and what are its known biological functions?

WDR66 is a protein containing 9 highly conserved WD40 repeat motifs and an EF-hand-like domain. It functions mechanically in cellular processes involving protein-protein interactions . Current research indicates it plays significant roles in:

• Spermatozoa motility regulation

• Cilium motility regulation through assembly of axonemal radial spokes

• Potential involvement in epithelial-mesenchymal transition (EMT) in cancer cells

WDR66 belongs to the family of WD-repeat proteins, several of which have been identified as tumor markers frequently up-regulated in various cancers . Among normal human tissues, WDR66 is most abundantly expressed in testis, suggesting it may function as a cancer/testis antigen .

Why is WDR66 significant in cancer research?

WDR66 has emerged as a potentially valuable biomarker in cancer research for several reasons:

These findings position WDR66 as both a potential prognostic marker and therapeutic target, particularly for esophageal squamous cell carcinoma.

What types of WDR66 antibodies are available for research applications?

Several types of WDR66 antibodies are currently available for research:

• Mouse Monoclonal Antibodies:

  • Example: Anti-WDR66 antibody [2A6F7] (ab175369)

  • Applications: Western Blot, Flow Cytometry

  • Reactivity: Human

  • Immunogen: Recombinant Fragment Protein within Human CFAP251 aa 1-250

• Mouse Polyclonal Antibodies:

  • Example: WDR66 Antibody (H00144406-B01P)

  • Applications: Western Blot, Immunocytochemistry/Immunofluorescence

  • Reactivity: Human

  • Format: Azide and BSA Free

When selecting an antibody, researchers should consider their specific application needs, the region of WDR66 being targeted, and validated reactivity in their experimental system.

How does WDR66 expression influence oncogenic pathways in esophageal squamous cell carcinoma?

WDR66 appears to influence multiple oncogenic pathways in ESCC:

• EMT Modulation: WDR66 knockdown studies revealed significant changes in EMT markers. Specifically:

  • Vimentin expression was downregulated upon WDR66 knockdown

  • Occludin, a tight junction protein, was markedly upregulated

  These findings suggest WDR66 positively modulates EMT, a critical process in cancer invasion and metastasis.

• Cell Proliferation: siRNA-mediated knockdown of WDR66 resulted in suppression of cell growth , indicating a role in promoting cancer cell proliferation.

• Cell Motility: WDR66 knockdown reduced cell motility , suggesting involvement in metastatic potential.

• Membrane Component Regulation: WDR66 knockdown particularly affected expression of membrane components , pointing to potential roles in cell-cell interactions and signaling.

The exact molecular mechanisms by which WDR66 influences these pathways remain areas for further investigation, particularly regarding its protein interaction partners and downstream signaling effects.

What is the relationship between WDR66 expression and clinical parameters in cancer patients?

Analysis of WDR66 expression in relation to clinicopathological parameters has revealed several significant associations:

• Survival Outcome: High expression of WDR66 RNA is a significant prognostic factor for poor cancer-related survival (P = 0.031) .

• Correlation with Clinical Parameters:

  • Significant association with tumor differentiation (P = 0.032)

  • Significant association with TNM stage (P = 0.002)

  • No significant association with gender (P = 0.804), age (P = 0.432), pT factor (P = 0.234), lymph node metastasis (P = 0.545), or distant metastasis (P = 0.543)

• Independent Prognostic Value: Multivariate Cox regression analysis confirmed that WDR66 expression remains an independent prognostic factor (P = 0.042) , suggesting its utility as a biomarker independent of other clinical parameters.

These correlations highlight the potential clinical utility of WDR66 expression analysis in risk stratification for ESCC patients, potentially guiding treatment decisions and follow-up protocols.

How can researchers differentiate between WDR66 and other WD-repeat proteins in experimental studies?

Differentiating WDR66 from other WD-repeat proteins requires careful experimental design:

• Antibody Selection: Use antibodies targeting unique regions of WDR66 outside the conserved WD40 domains to minimize cross-reactivity. The EF-hand-like domain of WDR66 may serve as a distinctive target .

• Validation Controls:

  • Include both positive controls (WDR66-transfected cell lysates) and negative controls (non-transfected lysates)

  • Use WDR66 recombinant protein as standard

  • For siRNA experiments, include scrambled siRNA controls to confirm specificity of knockdown effects

• Multiple Detection Methods: Combine protein detection (Western blot, immunofluorescence) with nucleic acid-based approaches (qRT-PCR, in situ hybridization) to confirm specificity .

• Expression Pattern Analysis: WDR66 shows distinctive tissue expression patterns (high in testis among normal tissues, specifically elevated in ESCC among cancer types) that can help confirm target identity .

• Molecular Weight Verification: Confirm the expected molecular weight of the detected protein (WDR66 predicted band size: 131 kDa, though some antibodies detect a 53.9 kDa band) .

What are optimal protocols for detecting WDR66 expression in tissue samples?

Several complementary approaches can be used for optimal WDR66 detection in tissue samples:

• Quantitative Real-Time PCR (qRT-PCR):

  • Successfully used to validate WDR66 expression differences between ESCC and normal tissues

  • Can establish cutoff values for prognostic stratification (e.g., WDR66 ≤ 125: low; WDR66 > 125: high)

  • Requires careful primer design to ensure specificity for WDR66

• In Situ Hybridization:

  • Effective for cellular localization of WDR66 mRNA in tissue sections

  • Protocol example: Use 148 bp fragment from 3' terminal end of human WDR66 gene (NM144668)

  • Label probe with digoxigenin/dUTP and detect using alkaline phosphatase method

  • Enables distinction between tumor cells and surrounding stroma

• Immunohistochemistry/Immunofluorescence:

  • Select antibodies validated for tissue section applications

  • Include positive controls (known WDR66-expressing tissues) and negative controls

  • May require antigen retrieval optimization depending on fixation method

  • Consider using tissue microarrays for high-throughput analysis

• Western Blotting for Tissue Lysates:

  • Recommended dilutions: 1:500 for antibodies like ab175369

  • Include appropriate controls (non-transfected vs. WDR66-transfected lysates)

  • Confirm expected molecular weight (131 kDa predicted, though variations may occur)

What strategies should be employed for WDR66 knockdown studies?

Based on published research, effective WDR66 knockdown studies should consider:

• siRNA Design and Delivery:

  • Validated siRNA sequence: 5' – GuuACuAAAGGuGAGCAuA - 3'

  • Optimal concentration: 25 nmol/L

  • Transfection reagents: Thermo Scientific DharmaFECT or equivalent

• Cell Line Selection:

  • KYSE520 (human esophageal squamous cell carcinoma line) shows high endogenous WDR66 expression

  • Most other cell lines (OE33, SW480, HT29, HCT116, LS174T, Caco2, HL60, HEK293, Daudi, Capan1, MCF7, MDA-MB231, MDA-MB435) show minimal expression

• Validation of Knockdown Efficiency:

  • qRT-PCR to confirm reduction in mRNA levels

  • Western blotting to confirm reduction in protein levels

  • Include time-course analysis to determine optimal post-transfection timepoint

• Downstream Analysis:

  • Microarray analysis to identify global gene expression changes (as performed in reference study)

  • Western blotting for specific targets (e.g., vimentin, occludin)

  • Functional assays: cell proliferation, motility, wound healing

• Statistical Analysis:

  • Nonparametric Mann–Whitney U test for cell numbers, motility assay, and cell wound assay after knockdown

  • Multiple biological replicates to ensure reproducibility

How can researchers validate the specificity of WDR66 antibodies?

Rigorous validation of WDR66 antibody specificity is critical for reliable research outcomes:

• Western Blot Validation:

  • Compare bands from non-transfected controls versus WDR66-transfected cell lysates

  • Include recombinant WDR66 protein as positive control

  • Verify expected molecular weight (predicted: 131 kDa, though some antibodies detect a 53.9 kDa band)

  • Test antibody performance across different cell lines with known WDR66 expression levels

• Knockdown Confirmation:

  • Perform siRNA-mediated knockdown of WDR66

  • Confirm reduction in band intensity by Western blot

  • Include scrambled siRNA as negative control

• Cross-Reactivity Assessment:

  • Test antibody against other WD-repeat proteins to ensure specificity

  • Consider orthogonal methods (mass spectrometry) to confirm antibody target identity

• Application-Specific Validation:

  • For Flow Cytometry: Include isotype controls and blocking experiments

  • For Immunocytochemistry: Include peptide competition assays

  • For each application, optimize antibody concentration (typical starting dilutions: 1:200 for flow cytometry, 1:500 for Western blot)

How should researchers design experiments to investigate WDR66's role in epithelial-mesenchymal transition?

Given WDR66's apparent role in EMT regulation, the following experimental design is recommended:

• Expression Correlation Studies:

  • Analyze correlation between WDR66 expression and established EMT markers (E-cadherin, vimentin, occludin, etc.) in tissue samples

  • Use both RNA-seq/qRT-PCR and protein-level methods

  • Consider single-cell approaches to address tumor heterogeneity

• Gain and Loss of Function Experiments:

  • WDR66 knockdown using validated siRNA sequences (5' – GuuACuAAAGGuGAGCAuA - 3')

  • WDR66 overexpression using expression vectors

  • Assess changes in EMT markers at both RNA and protein levels

  • Monitor morphological changes associated with EMT

• Functional Assays:

  • Migration assays (transwell, wound healing)

  • Invasion assays (Matrigel)

  • Cell adhesion assays

  • Measure markers of cell motility and invasiveness

• Molecular Mechanism Investigation:

  • Co-immunoprecipitation to identify WDR66 protein interaction partners

  • Chromatin immunoprecipitation if transcriptional regulation is suspected

  • Subcellular localization studies using cell fractionation and immunofluorescence

• In vivo Validation:

  • Xenograft models with WDR66 modulation

  • Assess tumor growth, invasion, and metastasis

  • Correlate with EMT marker expression in tumor tissues

What approaches can resolve inconsistent WDR66 antibody performance across different samples?

When encountering variable antibody performance, consider implementing these troubleshooting strategies:

• Sample Preparation Optimization:

  • Test different protein extraction methods (RIPA, NP-40, urea-based buffers)

  • Evaluate fresh vs. frozen samples vs. FFPE specimens

  • Add protease inhibitors to prevent degradation

  • Consider phosphatase inhibitors if phosphorylation affects antibody binding

• Antibody Selection and Handling:

  • Test multiple antibodies targeting different epitopes of WDR66

  • Compare monoclonal vs. polyclonal antibodies

  • Prepare fresh dilutions of antibody before each experiment

  • Validate antibody lot-to-lot consistency

• Protocol Modifications:

  • Adjust blocking conditions (BSA vs. milk, concentration, duration)

  • Optimize antibody concentration and incubation conditions

  • Test different detection systems (ECL vs. fluorescent)

  • For immunohistochemistry/immunofluorescence, test multiple antigen retrieval methods

• Control Implementation:

  • Include positive controls (WDR66-expressing tissue/cells)

  • Include negative controls (tissues with low/no expression)

  • Use recombinant WDR66 protein as standard

  • Consider spike-in experiments with recombinant protein

• Alternative Detection Methods:

  • Complement protein detection with mRNA-based methods (qRT-PCR, in situ hybridization)

  • Consider mass spectrometry for protein identification and quantification

  • Use genetic tagging approaches (FLAG, HA) for recombinant expression systems

How can researchers effectively analyze WDR66 expression data for clinical correlations?

When analyzing WDR66 expression data for clinical correlations, researchers should implement these methodological approaches:

• Expression Cut-point Determination:

  • Use maximally selected rank statistics to determine optimal value for separating groups

  • Example from literature: WDR66 ≤ 125: low; WDR66 > 125: high

  • Alternatively, use median or quartile-based stratification

• Statistical Methods for Survival Analysis:

  • Kaplan-Meier survival analysis with log-rank statistics

  • Cox proportional hazards regression for multivariate analysis

  • Include relevant clinical covariates (TNM stage, tumor differentiation, etc.)

• Correlation with Clinical Parameters:

  • Use appropriate statistical tests based on data distribution:

    • t-test with Welch's correction for qRT-PCR measurements

    • Nonparametric tests for non-normally distributed data

  • Analyze correlations with TNM stage, differentiation, metastasis, etc.

• Software Tools:

  • GraphPad Prism for initial statistical analysis and visualization

  • SPSS for comprehensive multivariate analysis

  • R packages for advanced biostatistical analysis

• Validation Approaches:

  • Split cohort into discovery and validation sets

  • Use independent cohorts for external validation

  • Implement cross-validation techniques

  • Consider meta-analysis of multiple studies

What are promising areas for further investigation of WDR66 in cancer biology?

Based on current knowledge, several promising research directions for WDR66 include:

• Functional Mechanisms:

  • Detailed protein interaction network of WDR66 in normal and cancer cells

  • Structural studies of WDR66's WD40 domains and EF-hand-like domain

  • Post-translational modifications affecting WDR66 function

• Clinical Applications:

  • Multimarker panels incorporating WDR66 for improved prognostication

  • Development of targeted therapies against WDR66 or its downstream effectors

  • Liquid biopsy approaches for monitoring WDR66 expression

• Role in Other Cancers:

  • Expanding investigation beyond ESCC to other cancer types

  • Comparative analysis across different tumor types

  • Pan-cancer bioinformatic analysis of WDR66 expression and mutations

• Relationship to Cilia Function:

  • Investigating the intersection between WDR66's role in cilia/flagella and cancer

  • Exploring potential connections to ciliopathies

  • Examining the evolutionary conservation of WDR66 functions

• Therapeutic Targeting:

  • Development of small molecule inhibitors of WDR66

  • Investigation of synthetic lethality approaches

  • Evaluation of WDR66 as an immunotherapy target (given its cancer/testis antigen properties)