EYA1 Antibody

What is EYA1 and what are its key biological functions?

EYA1 (Eyes Absent Homolog 1) is a transcriptional coactivator and protein tyrosine phosphatase with a molecular weight of approximately 64.6 kilodaltons in humans . It functions as a critical developmental regulator and is also known by alternative names including BOP, BOR, BOS1, and OFC1 . EYA1 plays essential roles in organogenesis during embryonic development, particularly in eye, ear, and kidney formation. In pathological contexts, EYA1 has been identified as a regulator in cancer stem cells, notably in glioblastoma where it interacts with MYC to influence tumor cell proliferation, migration, and self-renewal . The dual functionality of EYA1 as both a phosphatase and transcriptional regulator makes it a complex and important target for research across developmental biology, cancer biology, and regenerative medicine fields.

What types of EYA1 antibodies are currently available for research applications?

Research-grade EYA1 antibodies are available in multiple formats with varying specificity and applications:

Many suppliers offer antibodies targeting different epitopes of EYA1, including N-terminal, C-terminal, and middle region-specific antibodies . Some antibodies demonstrate cross-reactivity with other EYA family members (particularly EYA4), which should be considered when designing experiments requiring high specificity . Multiple species-reactive antibodies allow for comparative studies across model organisms, with human, mouse, and rat being the most commonly available reactivities.

How should I select the appropriate EYA1 antibody for my specific experimental application?

Selection of the appropriate EYA1 antibody requires careful consideration of several experimental factors:

For co-immunoprecipitation studies examining EYA1-MYC interactions, antibodies validated for immunoprecipitation that target regions not involved in the interaction would be optimal .

What are optimal conditions for using EYA1 antibodies in Western blotting experiments?

For successful Western blotting with EYA1 antibodies, consider these protocol optimizations:

• Sample preparation:

  • Use RIPA or IP lysis buffer containing protease and phosphatase inhibitors

  • Denature samples at 95°C for 5 minutes in reducing conditions

  • Load 20-40μg of total protein for cell lysates; 50-100μg for tissue samples

• Gel selection and transfer:

  • Use 8-10% SDS-PAGE gels due to EYA1's 64.6 kDa size

  • Transfer to PVDF membranes at 100V for 90 minutes (wet transfer) or 25V for 10 minutes (semi-dry)

• Blocking and antibody incubation:

  • Block with 5% non-fat dry milk in TBST for 1 hour at room temperature

  • Dilute primary antibodies 1:500-1:2000 depending on manufacturer recommendations

  • Incubate with primary antibody overnight at 4°C

  • Use HRP-conjugated secondary antibodies at 1:5000-1:10000 dilution

• Detection considerations:

  • The expected band size for human EYA1 is approximately 65 kDa

  • Verify specificity using positive controls (tissues/cells known to express EYA1)

  • Include negative controls such as knockdown samples when possible

When analyzing multiple EYA family members, perform sequential probing with appropriate stripping between antibodies to avoid cross-reactivity issues.

How can I design effective EYA1 co-immunoprecipitation experiments?

Co-immunoprecipitation (Co-IP) is valuable for studying EYA1 protein interactions, such as the EYA1-MYC axis in glioblastoma stem cells . For successful EYA1 Co-IP:

• Cell preparation:

  • Use approximately 1×10^7 cells per condition

  • Wash cells twice with cold PBS before lysis

  • Lyse cells in 1ml cold IP lysis buffer containing protease and phosphatase inhibitors

  • Incubate at 4°C for 30 minutes with occasional vortexing

• Pre-clearing and immunoprecipitation:

  • Pre-clear lysates with protein A/G mixtures to reduce non-specific binding

  • Incubate pre-cleared lysates with anti-EYA1 antibody at 4°C for 2 hours with rotation

  • Add 50μl of protein A/G beads in pre-chilled lysis buffer and incubate at 4°C overnight

• Washing and elution:

  • Wash immunoprecipitates 4-5 times with cold IP buffer

  • Elute bound proteins by boiling in SDS sample buffer for 5 minutes

• Analysis and controls:

  • Include IgG control immunoprecipitations

  • Confirm interactions by reciprocal Co-IP (e.g., IP with anti-MYC and blot for EYA1)

  • Verify protein interactions by alternative methods (e.g., proximity ligation assay)

This experimental approach has successfully demonstrated EYA1-MYC binding in glioblastoma stem cells, revealing a potential regulatory mechanism .

How can I use EYA1 antibodies to investigate EYA1's role in cancer stem cells?

EYA1 has been identified as preferentially expressed in glioblastoma stem cells (GSCs) compared to differentiated tumor progeny . To investigate EYA1's role in cancer stem cell biology:

• Comparative expression analysis:

  • Use immunofluorescence with EYA1 antibodies to compare expression between stem-like and differentiated cancer cell populations

  • Co-stain with stem cell markers (CD133, SOX2, NESTIN) to correlate EYA1 expression with stemness

• Functional studies with EYA1 knockdown:

  • Design lentiviral shRNA constructs targeting EYA1 (e.g., using validated sequences like TRCN0000369592, TRCN0000310704, or TRCN0000303462)

  • Confirm knockdown efficiency by Western blotting with EYA1 antibodies

  • Assess effects on stem cell properties: sphere formation, expression of stemness markers, multilineage differentiation potential

• Regulatory network analysis:

  • Investigate EYA1-MYC interaction through co-immunoprecipitation with EYA1 antibodies

  • Examine how EYA1 modulation affects MYC levels and activity

  • Perform ChIP-seq with EYA1 antibodies to identify genomic binding sites in stem versus differentiated cells

• In vivo validation:

  • Use EYA1 antibodies for immunohistochemical analysis of patient-derived xenografts

  • Compare EYA1 expression in tumor-initiating regions versus bulk tumor

This multifaceted approach can help elucidate EYA1's role in maintaining the cancer stem cell phenotype and identify potential therapeutic vulnerabilities, as demonstrated in glioblastoma research .

What methods can be used to study EYA1's phosphatase activity versus its transcriptional co-activator function?

EYA1 possesses dual functionality as both a tyrosine phosphatase and transcriptional co-activator. Distinguishing between these functions requires specialized experimental approaches:

• Phosphatase activity assays:

  • Immunoprecipitate EYA1 using specific antibodies and assess phosphatase activity with artificial substrates (p-nitrophenyl phosphate) or physiological substrates

  • Design domain-specific mutants (e.g., D327A mutation in the phosphatase domain) that selectively inhibit phosphatase function while preserving co-activator function

  • Use phosphatase-specific inhibitors to distinguish activity-dependent functions

• Transcriptional co-activator function:

  • Perform ChIP assays using EYA1 antibodies to identify genomic binding sites

  • Use reporter gene assays with EYA1 binding sites to quantify transcriptional activation

  • Test co-immunoprecipitation with known transcriptional partners (SIX family proteins)

• Dissecting dual functionality:

  • Use domain-specific antibodies that selectively recognize either the phosphatase domain or regions involved in transcriptional activation

  • Compare phospho-protein profiles using phospho-specific antibodies in cells with wild-type EYA1 versus phosphatase-dead mutants

  • Perform differential gene expression analysis after selectively inhibiting each function

• Novel interaction studies:

  • Investigate the EYA1-MYC interaction to determine whether it depends on phosphatase activity or transcriptional co-activation

  • Use proximity ligation assays with EYA1 antibodies to visualize protein interactions in situ

These approaches collectively provide a comprehensive assessment of EYA1's dual functionality in experimental models.

How can I design rigorous EYA1 knockdown validation experiments?

Rigorous validation of EYA1 knockdown is essential for interpreting phenotypic effects. A comprehensive validation strategy includes:

• Multiple shRNA constructs:

  • Use at least three non-overlapping shRNA sequences targeting different regions of EYA1 mRNA (e.g., validated sequences TRCN0000369592, TRCN0000310704, and TRCN0000303462)

  • Include non-targeting shRNA controls (e.g., SHC002)

• Multi-level validation:

  • mRNA level: Quantitative RT-PCR to measure EYA1 transcript reduction

  • Protein level: Western blotting with EYA1 antibodies to confirm protein depletion

  • Functional level: Assays to measure known EYA1-dependent activities

• Specificity controls:

  • Assess expression of other EYA family members (EYA2-4) to confirm specificity

  • Test for rescue of phenotypes by expressing shRNA-resistant EYA1 constructs

  • Compare phenotypes across multiple shRNAs to distinguish on-target from off-target effects

• Temporal considerations:

  • Establish time course of knockdown to correlate with phenotypic changes

  • Consider inducible knockdown systems for studying developmental processes

• Documentation requirements:

  • Image entire Western blots with molecular weight markers

  • Quantify knockdown efficiency across biological replicates

  • Document all experimental conditions in detail for reproducibility

This comprehensive validation approach ensures that observed phenotypes can be confidently attributed to EYA1 depletion rather than off-target effects.

What are common technical issues when using EYA1 antibodies and how can they be resolved?

Researchers frequently encounter several challenges when working with EYA1 antibodies:

• High background in immunostaining:

  • Problem: Non-specific staining obscuring true EYA1 signal

  • Solutions: Increase blocking time/concentration; use alternative blocking agents (BSA, serum, commercial blockers); optimize antibody dilution; include additional washing steps; use monoclonal antibodies for greater specificity

• Weak or absent signal in Western blots:

  • Problem: Inability to detect EYA1 despite expected expression

  • Solutions: Optimize protein extraction method for nuclear proteins; increase protein loading; reduce transfer time for large proteins; use enhanced detection systems; verify sample preparation preserves EYA1 integrity; try antibodies targeting different epitopes

• Multiple bands in Western blots:

  • Problem: Unclear which band represents genuine EYA1

  • Solutions: Include positive control samples; perform EYA1 knockdown to identify specific bands; consult literature for expected band patterns; consider post-translational modifications or isoforms

• Inconsistent immunoprecipitation results:

  • Problem: Variable or poor pull-down efficiency

  • Solutions: Optimize lysis conditions to preserve protein interactions; pre-clear lysates thoroughly; extend antibody incubation time; use crosslinking approaches for transient interactions; test different antibody-bead combinations

• Cross-reactivity with other EYA proteins:

  • Problem: Antibody recognizes multiple EYA family members

  • Solutions: Verify antibody specificity through knockout/knockdown controls; use epitope-specific antibodies; perform comparative analysis with multiple antibodies; consider computational sequence analysis to identify unique epitopes

Each troubleshooting approach should be systematically documented to establish optimal conditions for your specific experimental system.

How should I interpret conflicting results from different EYA1 antibodies?

Conflicting results from different EYA1 antibodies are not uncommon and require careful interpretation:

• Epitope accessibility analysis:

  • Different antibodies recognize distinct epitopes that may be differentially accessible in various experimental conditions

  • Map the recognized epitopes to functional domains of EYA1 and consider whether protein interactions or conformational changes might mask specific regions

  • Test multiple antibodies recognizing different regions in parallel

• Cross-reactivity assessment:

  • Evaluate whether discrepancies result from differential recognition of EYA family members

  • Perform side-by-side validation in systems with known EYA1 expression versus EYA2-4 expression

  • Use genetic approaches (siRNA, CRISPR) to validate antibody specificity

• Technical validation approaches:

  • Compare antibody performance across multiple lots and suppliers

  • Use recombinant EYA1 protein as a positive control

  • Validate key findings with orthogonal techniques not relying on antibodies (e.g., mass spectrometry)

• Contextual interpretation:

  • Consider that different results may reflect biological reality rather than technical artifacts

  • EYA1 undergoes post-translational modifications and exists in multiple complexes

  • Different antibodies may preferentially detect specific EYA1 subpopulations

• Resolution strategies:

  • For critical findings, confirm with at least two independent antibodies

  • Complement antibody-based methods with genetic approaches

  • Document all variables that might contribute to differential results

These analytical approaches transform potentially conflicting results into deeper insights about EYA1 biology and methodological considerations.

What statistical approaches are recommended when analyzing EYA1 expression data across different experimental conditions?

Robust statistical analysis of EYA1 expression data requires attention to several methodological considerations:

• Experimental design fundamentals:

  • Ensure adequate biological replicates (minimum n=3, preferably n≥5)

  • Include technical replicates to assess methodological variability

  • Design experiments to control for batch effects and confounding variables

• Normalization strategies:

  • For Western blot quantification, normalize EYA1 signal to appropriate loading controls (GAPDH, β-actin, total protein)

  • In immunofluorescence, normalize to nuclear staining or cell area

  • For qRT-PCR, validate reference genes for stability across experimental conditions

• Statistical tests for different experimental scenarios:

  • Two-group comparisons: t-test (parametric) or Mann-Whitney (non-parametric)

  • Multiple group comparisons: ANOVA with appropriate post-hoc tests

  • Correlative studies: Pearson or Spearman correlation coefficients

  • Time-course experiments: Repeated measures ANOVA or mixed-effects models

• Advanced analytical approaches:

  • For heterogeneous samples (e.g., tumor specimens), consider multiparameter analysis correlating EYA1 with clinical variables

  • For complex datasets, employ multivariate analysis to identify patterns associated with EYA1 expression

  • Use machine learning approaches for predictive modeling based on EYA1 and related markers

• Reporting requirements:

  • Clearly state sample sizes, statistical tests, and p-values

  • Report effect sizes and confidence intervals, not just statistical significance

  • Include all data points in visualizations, not just means/medians

  • Document software and analysis parameters for reproducibility

How is EYA1 being investigated as a potential therapeutic target in cancer research?

EYA1 has emerged as a promising therapeutic target, particularly in cancer stem cell biology:

• Glioblastoma stem cell targeting:

  • EYA1 is preferentially expressed in glioblastoma stem cells compared to differentiated tumor progeny

  • Targeting EYA1 decreases GSC proliferation, migration, and self-renewal in vitro and reduces tumor growth in vivo

  • The EYA1-MYC interaction represents a potential therapeutic vulnerability, as EYA1 targeting leads to loss of MYC expression

• Therapeutic development approaches:

  • Small molecule inhibitors: Targeting EYA1's phosphatase domain

  • Peptide-based inhibitors: Disrupting protein-protein interactions, particularly with SIX proteins or MYC

  • Gene therapy approaches: Using shRNA or CRISPR systems for EYA1 inhibition in tumors

• Targeting strategies:

  • Direct targeting of EYA1 activity or expression

  • Indirect targeting of downstream pathways

  • Combination approaches targeting both EYA1 and interacting partners like MYC

• Biomarker potential:

  • EYA1 expression as a prognostic or predictive biomarker

  • EYA1 activity as a measure of therapeutic response

The emerging understanding of EYA1's role in regulating stem-like properties in tumors, particularly through interaction with MYC, provides a compelling rationale for continued investigation as a therapeutic target .

What are the latest methodologies for studying EYA1 in developmental contexts?

Recent advances have enhanced our ability to study EYA1's developmental functions:

• Advanced imaging techniques:

  • Live imaging using fluorescently-tagged EYA1 to track subcellular localization during development

  • Super-resolution microscopy to visualize EYA1 interactions with nuclear components

  • Intravital imaging of EYA1 expression in developing organs in model organisms

• Genetic engineering approaches:

  • CRISPR/Cas9-mediated genome editing to introduce specific mutations in EYA1

  • Conditional knockout models to study tissue-specific requirements

  • Creation of reporter lines where fluorescent proteins are expressed under endogenous EYA1 regulatory elements

• Single-cell techniques:

  • Single-cell RNA sequencing to identify EYA1-expressing populations during development

  • CyTOF and spectral flow cytometry to correlate EYA1 with developmental markers

  • Spatial transcriptomics to map EYA1 expression in developing tissues

• Organoid and 3D culture systems:

  • Using EYA1 antibodies to study expression and localization in organoids

  • Assessing effects of EYA1 manipulation on organoid formation and differentiation

  • Co-culture systems to study cell-cell interactions mediated by EYA1

These methodologies allow researchers to address fundamental questions about EYA1's role in organ development and morphogenesis with unprecedented spatial and temporal resolution.

What novel insights have emerged about EYA1's role in regulating signaling pathways?

Recent research has expanded our understanding of EYA1's regulatory networks:

• EYA1-MYC regulatory axis:

  • EYA1 physically interacts with MYC in glioblastoma stem cells

  • Targeting EYA1 expression causes loss of MYC expression, suggesting a regulatory relationship

  • This interaction may represent a previously unrecognized mechanism for MYC regulation in stem-like cells

• Integrated pathway analysis:

  • Cross-talk between EYA1 and major developmental signaling pathways (Notch, Wnt, Sonic Hedgehog)

  • Integration of growth factor signaling with transcriptional control via EYA1

  • Combinatorial effects of EYA1 with SIX family members in target gene regulation

• Post-translational regulation:

  • Phosphorylation states affecting EYA1 function

  • Protein stability regulation through ubiquitin-proteasome system

  • Subcellular localization dynamics in response to cellular signals

• Phosphatase substrate identification:

  • Novel targets of EYA1's phosphatase activity

  • Functional consequences of substrate dephosphorylation

  • Differential substrate preferences in development versus disease contexts

These emerging insights highlight EYA1's position at the intersection of multiple regulatory networks, particularly in stem cell contexts where it may serve as a critical link between external signals and transcriptional responses through mechanisms including its newly identified interaction with MYC .