EYA3 Antibody

What is EYA3 and why is it significant in research?

EYA3 (Eyes Absent Homolog 3) is a critical developmental regulator that is highly expressed during embryogenesis but typically downregulated after development. EYA3 functions as both a transcriptional cofactor and a phosphatase with dual specificity, containing both threonine and tyrosine phosphatase activities. Its significance in research has grown substantially as studies have revealed its re-expression and amplification in multiple tumor types, particularly in breast cancer and Ewing sarcoma, where it plays roles in tumor progression through various mechanisms . EYA3's involvement in regulating immune responses in the tumor microenvironment has made it an emerging target for cancer research, particularly in understanding resistance to immunotherapy and the development of potential targeted treatments.

What are the common applications for EYA3 antibodies in research?

EYA3 antibodies are employed across multiple experimental techniques in research laboratories. The most frequent applications include Western Blot (WB) for protein expression quantification, Immunohistochemistry (IHC) for tissue localization, Immunofluorescence (IF) for cellular localization studies, Immunoprecipitation (IP) for protein-protein interaction investigations, and Flow Cytometry for quantitative cellular analysis . For Western Blotting, EYA3 antibodies typically detect a protein band at approximately 63-66 kDa, consistent with the predicted molecular weight of the protein. In IHC applications, these antibodies have been validated with human breast cancer tissue, making them particularly valuable for oncology research . Each application requires specific optimization of antibody dilution, with recommendations ranging from 1:500-1:1000 for WB to 1:50-1:500 for IHC protocols.

How should researchers select the appropriate EYA3 antibody for their specific application?

Selection of the optimal EYA3 antibody depends critically on both the experimental application and target species. For human samples, commercially available antibodies like the polyclonal 21196-1-AP have demonstrated reactivity across multiple techniques including WB, IHC, IF/ICC, IP, and ELISA . When designing experiments, researchers should consider several factors: (1) Reactivity spectrum - confirm the antibody has been validated for your species of interest; (2) Application compatibility - verify successful use in your specific technique through literature or validation data; (3) Clonality - polyclonal antibodies offer higher sensitivity but potentially lower specificity compared to monoclonals; (4) Detection method compatibility - ensure the antibody works with your visualization system; and (5) Reproducibility considerations - check if the antibody has been used in published research. For applications requiring quantitative analysis of EYA3 expression in breast cancer or Ewing sarcoma tissues, antibodies validated specifically in these contexts will yield more reliable results.

How can EYA3 antibodies help investigate its role in tumor-associated immune suppression?

EYA3 antibodies serve as critical tools for elucidating the complex role of EYA3 in tumor-associated immune suppression, particularly in triple-negative breast cancer (TNBC). Research has established that EYA3 employs its threonine phosphatase activity to dephosphorylate Myc at the pT58 position, leading to Myc stabilization and subsequent upregulation of PD-L1, a key immunosuppressive molecule . To investigate this mechanism, researchers can employ EYA3 antibodies in multiplex immunofluorescence approaches to simultaneously visualize EYA3, PD-L1, and immune cell markers like CD8 within the tumor microenvironment. This approach allows for spatial correlation analysis between EYA3 expression and infiltrating CD8+ T cells. Additionally, chromatin immunoprecipitation (ChIP) experiments using EYA3 antibodies can help identify the genomic regions where EYA3 functions as a transcriptional cofactor, further clarifying its role in immune regulation. When designing such experiments, researchers should account for the significant correlation observed between EYA3 and PD-L1 expression in human breast tumors and consider dual staining approaches to verify coexpression patterns .

What methodological considerations are important when studying EYA3's relationship to chemoresistance in cancer?

When investigating EYA3's contribution to chemoresistance mechanisms, researchers should implement a multi-faceted experimental approach. Studies in Ewing sarcoma have revealed that EYA3 knockdown sensitizes cells to DNA-damaging chemotherapeutics and impairs DNA damage repair . For robust methodological design, researchers should: (1) Establish appropriate cellular models with EYA3 manipulation (knockdown/overexpression) using verified antibodies to confirm alteration of protein levels; (2) Employ DNA damage assays (γH2AX foci detection, comet assay) before and after chemotherapeutic treatment while monitoring EYA3 expression with specific antibodies; (3) Perform time-course analyses of DNA repair efficiency using immunofluorescence double-staining with EYA3 and DNA damage markers; and (4) Validate findings across multiple cell lines and primary samples to ensure reproducibility. Importantly, researchers should account for the microRNA-mediated regulation of EYA3, particularly by miR-708, which has been shown to target the EYA3 3'-UTR in Ewing sarcoma . Correlation analyses between EYA3 protein levels (detected by antibody) and miR-708 expression can provide mechanistic insights into the regulatory network governing chemoresistance.

How can researchers use EYA3 antibodies to explore its dual phosphatase activities in cancer models?

EYA3 possesses unique dual phosphatase activities—threonine phosphatase and tyrosine phosphatase functions—that contribute differently to its oncogenic properties. To dissect these distinct enzymatic activities, researchers should design experiments that specifically target each function. For threonine phosphatase activity, which has been implicated in Myc stabilization through dephosphorylation at pT58 , researchers can use phospho-specific antibodies against Myc-pT58 alongside EYA3 antibodies in co-immunoprecipitation studies. Similarly, for tyrosine phosphatase activity analysis, researchers can employ phosphotyrosine-specific antibodies to examine global changes in tyrosine phosphorylation patterns following EYA3 manipulation. To distinguish between these activities experimentally, point mutations in EYA3's active sites can be introduced—specifically targeting residues required for threonine versus tyrosine phosphatase activities—followed by rescue experiments and functional readouts relevant to cancer progression. Immunofluorescence co-localization studies using EYA3 antibodies with phospho-substrate antibodies can provide spatial information about where within the cell these different phosphatase activities predominate, offering insights into subcellular mechanisms of action.

What are the optimal protocols for using EYA3 antibodies in immunohistochemistry of cancer tissues?

For successful immunohistochemical detection of EYA3 in cancer tissues, researchers should adhere to an optimized protocol that accounts for the protein's expression characteristics and tissue-specific factors. Based on validated methodologies, the following approach is recommended: Begin with formalin-fixed, paraffin-embedded tissue sections of 4-5μm thickness. For antigen retrieval, use TE buffer at pH 9.0, although citrate buffer at pH 6.0 may serve as an acceptable alternative . When working with breast cancer tissues, where EYA3 expression has been specifically validated, use the EYA3 antibody at a dilution range of 1:50-1:500, optimizing the exact dilution for each specific antibody lot and tissue type . For visualization, a polymer-based detection system with DAB as chromogen typically provides strong signal-to-noise ratio. Include positive controls (HeLa cells or known EYA3-expressing breast cancer tissues) and negative controls (primary antibody omission) in each staining run to verify specificity. When analyzing results, researchers should score both nuclear and cytoplasmic EYA3 staining separately, as the protein's function can differ based on subcellular localization. For correlation with immune parameters, consider using serial sections with CD8 and PD-L1 staining to investigate relationships between EYA3 expression and the immune microenvironment .

What controls and validation steps are necessary when using EYA3 antibodies in research?

Rigorous validation of EYA3 antibodies is essential for generating reliable and reproducible research outcomes. A comprehensive validation strategy should include: (1) Positive and negative cellular controls—use cell lines with confirmed EYA3 expression (e.g., HeLa, L02) as positive controls and EYA3 knockdown/knockout cells as negative controls ; (2) Peptide competition assays—pre-incubation of the antibody with its immunizing peptide should abolish specific signals; (3) Cross-validation with multiple detection methods—confirm findings using at least two independent techniques (e.g., WB and IHC); (4) Specificity verification through siRNA or CRISPR-mediated EYA3 depletion—demonstrate signal reduction proportional to knockdown efficiency; (5) Orthogonal validation—compare results with alternative antibodies targeting different EYA3 epitopes; and (6) Validation across relevant experimental models—test antibody performance in all specific tissue types and experimental conditions planned for the study. For cancer research applications, researchers should additionally validate antibody performance in the specific tumor types of interest, as EYA3 expression patterns and potential post-translational modifications may vary between cancer types .

How should researchers optimize Western blot protocols for EYA3 detection?

For optimal detection of EYA3 via Western blot, researchers should implement a tailored protocol that addresses the specific characteristics of this 63-66 kDa protein. Sample preparation should begin with efficient lysis using RIPA buffer supplemented with protease and phosphatase inhibitors, particularly important when studying EYA3's phosphatase functions or phosphorylation status. For gel electrophoresis, 8-10% polyacrylamide gels provide optimal resolution for the EYA3 molecular weight range. During transfer to nitrocellulose or PVDF membranes, an overnight transfer at lower voltage (30V) at 4°C often yields better results for larger proteins like EYA3. For blocking, 5% non-fat milk in TBST is typically effective, though for phospho-specific applications, 5% BSA may be preferable. The recommended antibody dilution range is 1:500-1:1000 , but optimization through dilution series is advised for each experimental system. When developing the blot, extended exposure times may be necessary if EYA3 expression is low in certain cell types. For quantitative analysis, researchers should normalize EYA3 signals to appropriate loading controls and perform at least three biological replicates. When studying cancer samples, particularly breast cancer or Ewing sarcoma, researchers should be aware that EYA3 may show variable expression levels across different molecular subtypes .

How can researchers address inconsistent EYA3 antibody staining patterns in immunohistochemistry?

Inconsistent EYA3 staining patterns in immunohistochemistry can stem from multiple technical and biological factors. To systematically address this issue, researchers should first examine fixation conditions, as overfixation or underfixation can significantly impact antibody accessibility to epitopes. The choice of antigen retrieval method is particularly critical—for EYA3, TE buffer at pH 9.0 is recommended as the primary approach, with citrate buffer at pH 6.0 as an alternative . Researchers experiencing inconsistent results should compare both methods. Antibody concentration requires careful titration; testing a dilution series from 1:50 to 1:500 can identify the optimal working concentration for specific tissue types . The detection system employed (streptavidin-biotin versus polymer-based) can also impact staining consistency. From a biological perspective, researchers should recognize that EYA3 exhibits both nuclear and cytoplasmic localization, with patterns potentially varying across tissue types and cancer subtypes. When evaluating breast cancer specimens specifically, researchers should note that EYA3 expression is typically higher in triple-negative breast cancer compared to other subtypes, which might explain apparent staining inconsistencies across unclassified samples . Additionally, EYA3's expression in immune cells within the tumor microenvironment may complicate interpretation, necessitating dual staining approaches to distinguish tumor cell expression from immune cell expression.

What are the common pitfalls in interpreting EYA3 expression data in cancer research?

Interpretation of EYA3 expression data in cancer research requires awareness of several potential pitfalls. First, researchers must recognize the context-dependent nature of EYA3 function—while it promotes tumor progression in breast cancer through immune suppression mechanisms , its effects may differ in other cancer types or cellular contexts. Second, subcellular localization significantly impacts EYA3's function; therefore, analysis should distinguish between nuclear and cytoplasmic expression rather than relying solely on total protein levels. Third, EYA3's relationship with the immune microenvironment creates complexity—high EYA3 expression correlates with decreased CD8+ T cell infiltration in breast tumors , but this relationship may vary by cancer type or treatment status. Fourth, researchers must account for the regulatory mechanisms controlling EYA3 expression, including miRNA-mediated regulation (e.g., miR-708 in Ewing sarcoma) , when interpreting expression patterns. Fifth, the dual phosphatase activities of EYA3 (threonine and tyrosine phosphatase) contribute differently to various cancer phenotypes, potentially confounding functional interpretations based solely on expression data. Finally, technical considerations like antibody cross-reactivity with other EYA family members (EYA1, EYA2, EYA4) should be evaluated when interpreting immunostaining or Western blot results. To mitigate these pitfalls, researchers should employ multiple detection methods, include appropriate controls, and integrate EYA3 expression data with functional assays.

How should researchers interpret contradictory findings regarding EYA3's role in different cancer types?

When faced with seemingly contradictory findings regarding EYA3's role across different cancer types, researchers should implement a systematic analytical framework. First, evaluate the cellular context—EYA3 functions as an oncogene in breast cancer and Ewing sarcoma , but its role may differ in other cancer types depending on the cellular background and molecular landscape. Second, consider EYA3's dual functionality—as both a transcriptional cofactor and a phosphatase with distinct substrates that may vary between cancer types. Third, examine the tumor microenvironment context—in breast cancer, EYA3 promotes immune suppression through PD-L1 upregulation , but this mechanism may not be uniformly relevant across all cancers. Fourth, analyze methodological differences between studies, including antibody specificity, detection methods, and experimental models. Fifth, consider genetic and epigenetic backgrounds—EYA3 is regulated by the EWS/FLI1 fusion protein in Ewing sarcoma through miR-708 repression , a mechanism unlikely to be present in cancers lacking this fusion. To reconcile contradictory findings, researchers should design comparative studies using standardized methodologies across multiple cancer types, employ both in vitro and in vivo models, and integrate multi-omics approaches to comprehensively assess EYA3's impact on different cancer phenotypes.

What are the recommended dilutions and applications for EYA3 antibodies in different experimental contexts?

The optimal utilization of EYA3 antibodies requires appropriate dilution adjustment based on the specific experimental technique. For Western blot applications, a dilution range of 1:500-1:1000 typically provides an optimal signal-to-noise ratio when detecting the 63-66 kDa EYA3 protein . Immunohistochemistry applications require more careful titration, with recommended dilutions ranging from 1:50-1:500 depending on tissue type and fixation conditions . For immunofluorescence and immunocytochemistry, a higher antibody concentration is typically needed, with suggested dilutions of 1:10-1:100 . Immunoprecipitation protocols should utilize 0.5-4.0 μg of antibody for every 1.0-3.0 mg of total protein lysate to achieve effective pull-down of EYA3 and its interacting partners . For flow cytometry applications focusing on intracellular EYA3 detection, approximately 0.25 μg of antibody per 10^6 cells in a 100 μl suspension is recommended . These parameters are summarized in the table below:

Researchers should note that these are starting recommendations, and antibody performance should be validated and optimized for each specific experimental system .

How can researchers design experiments to study EYA3's role in CD8+ T cell regulation in cancer?

To investigate EYA3's impact on CD8+ T cell regulation in cancer, researchers should design comprehensive experiments spanning in vitro, in vivo, and clinical specimen analyses. In vitro approaches should include co-culture systems where cancer cells with manipulated EYA3 expression (overexpression or knockdown) are cultured with isolated CD8+ T cells to assess functional parameters like T cell activation, exhaustion marker expression, and cytotoxic activity. For in vivo studies, researchers can employ orthotopic tumor models in immunocompetent mice using EYA3-manipulated cancer cell lines, followed by analysis of tumor growth kinetics, CD8+ T cell infiltration, and T cell phenotyping via flow cytometry . To assess the clinical relevance, patient tumor samples should be analyzed for correlations between EYA3 expression and CD8+ T cell infiltration using multiplex immunohistochemistry or immunofluorescence . Mechanistically, researchers should examine the EYA3-Myc-PD-L1 axis by quantifying Myc phosphorylation status at pT58 in relation to EYA3 expression levels, and validate findings through rescue experiments where PD-L1 is restored in EYA3-knockdown cells . Importantly, researchers should include CD8+ T cell depletion studies in mouse models to confirm the causal relationship between EYA3 expression and CD8+ T cell-mediated tumor control, as previous research has demonstrated that CD8+ T cell depletion reverses the growth inhibition observed in EYA3-knockdown tumors .