EYA4 Antibody

What is EYA4 and why is it important for research?

EYA4 is a member of the Eyes Absent (EYA) family of dual-functioning protein phosphatases. In humans, the canonical EYA4 protein consists of 639 amino acid residues with a molecular mass of 69.5 kDa. The protein localizes to both the nucleus and cytoplasm, with up to five different isoforms reported . EYA4 functions as a tyrosine phosphatase that specifically dephosphorylates 'Tyr-142' of histone H2AX (H2AXY142ph) and possesses serine/threonine phosphatase activity critical for cellular functions .

Research significance:

• Highly expressed in heart and skeletal muscle tissues

• Functions in both transcriptional activation and protein dephosphorylation

• Implicated in genome stability and DNA replication

• Plays critical roles in breast cancer progression and metastasis

• Can serve as a marker for specific neuronal populations including Cerebral Cortex MGE Interneurons and Hippocampal Gyrus Chandelier Neurons

What applications are EYA4 antibodies commonly used for?

EYA4 antibodies support multiple experimental applications for detecting and studying EYA4 protein. The most common applications include:

Researchers should validate specific dilutions for each antibody as recommendations may vary by manufacturer and experimental conditions .

What experimental controls should be included when using EYA4 antibodies?

Proper experimental controls are essential for obtaining reliable results with EYA4 antibodies:

• Positive control: Include samples known to express EYA4 (e.g., heart or skeletal muscle tissue, or cells transfected with EYA4 expression vectors)

• Negative control: Include samples with very low or no EYA4 expression (e.g., MCF-7 breast cancer cells express low or undetectable levels of endogenous EYA4)

• Knockdown/knockout control: Cells with shRNA-mediated EYA4 knockdown provide excellent specificity controls (e.g., using validated constructs like TRCN0000244430, TRCN0000218273, or TRCN0000244429)

• Secondary antibody control: Omit primary antibody to assess non-specific binding of secondary antibody

• Isotype control: Include appropriate isotype-matched control antibody to identify non-specific binding

• Peptide blocking: Pre-incubate antibody with immunizing peptide to confirm specificity

These controls help validate antibody specificity and ensure accurate interpretation of experimental results.

How can EYA4 antibodies be optimized for studying its role in DNA replication and genome stability?

EYA4 plays crucial roles in preventing genome instability by inhibiting replication-associated DNA damage. To effectively study these functions:

Recommended methodological approach:

• Replication stress assessment: Use EYA4 antibodies in combination with replication stress markers

  • Co-stain with γH2AX antibodies to detect DNA damage foci

  • Monitor ATR pathway activation using phospho-Chk1 (S345) antibodies

  • Assess replication fork progression through EdU incorporation assays

• Cell cycle analysis:

  • Combine EYA4 immunostaining with cell cycle markers like cyclin A, cyclin E1, and PCNA

  • Use flow cytometry to correlate EYA4 expression with cell cycle phases

  • Monitor CDK2, p21^WAF1/CIP1, and p27^KIP1 to assess cell cycle regulation

• Replication fork studies:

  • Use DNA fiber assays with EYA4 antibody immunoprecipitation to analyze proteins at replication forks

  • Apply hydroxyurea treatment (4mM) to induce replication stress followed by EdU labeling (10μM for 30 min)

  • Quantify EdU-positive cells using confocal microscopy and analysis software like CellProfiler

For optimal results, combine these approaches with EYA4 knockdown/overexpression systems to establish causal relationships between EYA4 expression and genomic stability phenotypes.

What are the methodological considerations when investigating EYA4's phosphatase activity?

EYA4 possesses dual phosphatase activity (tyrosine and serine/threonine), with its serine/threonine phosphatase domain being particularly important for cancer progression and replication fork dynamics .

Critical methodological considerations:

• Phosphatase domain-specific antibodies:

  • Use antibodies that recognize different domains of EYA4 (N-terminal vs. C-terminal)

  • Compare wild-type EYA4 with phosphatase-dead mutants (e.g., 3YF281 and pY dead mutants)

• Activity-based assays:

  • In vitro phosphatase assays using purified EYA4 protein and synthetic phosphopeptide substrates

  • Monitor dephosphorylation of histone H2AX at Tyr-142 as a functional readout

  • Assess phosphatase activity against serine/threonine substrates linked to replication regulation

• Phosphatase inhibitor studies:

  • Compare general phosphatase inhibitors with EYA-specific inhibitors

  • Analyze effects on downstream pathway components like ATR activation (measured by Chk1 phosphorylation)

  • Monitor replication stress markers following inhibitor treatment

A combined approach using phosphatase-specific antibodies, activity assays, and inhibitor studies provides comprehensive insights into EYA4's phosphatase functions in various cellular contexts.

How do you optimize detection protocols for low-abundance EYA4 protein?

When studying EYA4 in tissues or cell lines with low expression levels, standard detection methods may yield poor results. Advanced optimization techniques include:

• Signal amplification strategies:

  • Employ tyramide signal amplification (TSA) for immunohistochemistry and immunofluorescence

  • Use biotin-streptavidin systems to enhance detection sensitivity

  • Consider using conjugated antibodies (e.g., Cy5.5) for improved signal-to-noise ratio

• Immunoprecipitation before Western blotting:

  • Concentrate EYA4 protein by immunoprecipitation from large sample volumes

  • Use optimized lysis buffers containing phosphatase inhibitors (Na₃VO₄, NaF) to preserve phosphorylation status

  • Include protease inhibitors (PMSF, cOmplete Mini EDTA-free protease inhibitor cocktail) to prevent degradation

• Quantitative PCR validation:

  • Confirm protein expression data with qRT-PCR using TaqMan probes spanning across EYA4 exons

  • Use validated primers (e.g., Invitrogen Hs01012406_mH) with appropriate endogenous controls (e.g., 18S RNA, Invitrogen Hs99999901_s1)

  • Apply the 2^-ΔΔCt method for relative expression quantification

These approaches increase detection sensitivity while maintaining specificity, crucial for accurate assessment of EYA4 in research samples.

How do you address inconsistent Western blot results with EYA4 antibodies?

Inconsistent Western blot results are common challenges when working with EYA4 antibodies. Systematic troubleshooting approaches include:

Methodological solutions:

• Protein extraction optimization:

  • Use RIPA buffer supplemented with protease inhibitors, phosphatase inhibitors (1 mM Na₃VO₄, 1 mM NaF), and benzonase (0.025 U/μL)

  • Sonicate samples (2 min at 40% amplitude) to ensure complete lysis and DNA shearing

  • Standardize protein quantification methods and loading amounts (20-50 μg recommended)

• Membrane and blocking optimization:

  • Compare PVDF (Immobilon-P) versus nitrocellulose membranes

  • Test different blocking agents (5% skim milk versus 5% BSA in TBS-T)

  • Optimize primary antibody incubation (4°C overnight versus 2h at room temperature)

• Detection system considerations:

  • Compare HRP-conjugated versus fluorescent secondary antibodies

  • For weak signals, use enhanced chemiluminescent substrates (e.g., Clarity Western ECL)

  • Consider using gradient gels (4-20%) for better resolution of different EYA4 isoforms

• Antibody validation:

  • Test multiple EYA4 antibodies targeting different epitopes

  • Include positive controls (e.g., EYA4-overexpressing cells) and negative controls (e.g., EYA4 knockdown cells)

  • Consider checking for post-translational modifications that might affect antibody binding

What factors affect EYA4 antibody performance in immunohistochemistry applications?

IHC applications present unique challenges for EYA4 detection. Critical factors to consider include:

• Fixation and antigen retrieval:

  • Compare formalin-fixed paraffin-embedded (FFPE) versus frozen sections

  • Optimize antigen retrieval methods (citrate buffer pH 6.0 versus EDTA buffer pH 9.0)

  • Test different retrieval times and temperatures (microwave versus pressure cooker methods)

• Antibody selection and validation:

  • Validate antibodies specifically validated for IHC-p (paraffin) or IHC-fr (frozen) applications

  • Compare monoclonal versus polyclonal antibodies for specific applications

  • Verify antibody performance using positive control tissues (heart and skeletal muscle)

• Detection system optimization:

  • Compare direct detection versus amplification systems (ABC, polymer-based)

  • Optimize primary antibody concentration through serial dilutions

  • Reduce background by optimizing washing steps and using appropriate blocking reagents

Sample preparation quality significantly impacts IHC results, with factors such as tissue fixation time, processing protocols, and section thickness all influencing antibody performance.

How are EYA4 antibodies used to investigate its role in cancer progression?

EYA4 has been identified as a novel breast cancer oncogene that supports primary tumor growth and metastasis. Antibody-based approaches to investigate its role include:

• Expression profiling in cancer tissues:

  • IHC analysis of EYA4 expression across cancer stages and subtypes

  • Correlation of expression levels with clinical outcomes

  • Combination with other markers to develop prognostic signatures

• Functional studies in cancer models:

  • Analysis of EYA4 expression in cancer cell lines with varying metastatic potential

  • Monitoring changes in cellular phenotypes following EYA4 modulation

  • In vivo studies using tumor xenograft models with bioluminescence imaging

Research findings demonstrate that:

• EYA4 overexpression in MCF-7 cells (which normally express low EYA4) significantly increases tumor volume in mice

• Bioluminescence intensity measurements show enhanced tumor growth with EYA4 overexpression

• Tumors with EYA4 overexpression exhibit more aggressive histopathological features

These findings suggest EYA4 as a potential therapeutic target, with antibodies serving as crucial tools for detection, functional characterization, and possibly therapeutic development.

What methodological approaches can assess the impact of EYA4 on DNA damage and repair pathways?

EYA4's roles in DNA damage response and repair can be effectively investigated using antibody-based approaches:

• DNA damage assessment protocols:

  • Quantify γH2AX foci using immunofluorescence with EYA4 co-staining

  • Measure ATR pathway activation via phospho-Chk1 (S345) and phospho-Chk2 (T68) detection

  • Assess DNA damage accumulation in EYA4-depleted versus control cells

• Cell cycle checkpoint analysis:

  • Monitor cell cycle regulators (cyclin E1, CDK2, p21, p27, cyclin A) in EYA4-modulated cells

  • Use flow cytometry with EdU incorporation to assess S-phase progression

  • Analyze polyploidy resulting from endoreplication using DAPI staining and imaging

• Replication stress response:

  • Measure sensitivity to replication stress inducers (e.g., hydroxyurea) using MTT assays

  • Calculate cell viability using the formula: Cell viability (%) = (OD treated / OD control) × 100

  • Analyze EdU incorporation following hydroxyurea treatment (4mM for 2h) with 10-minute release

These methodological approaches provide mechanistic insights into how EYA4 contributes to genome stability and DNA damage response, with important implications for understanding its role in cancer progression.

How might EYA4 antibodies contribute to therapeutic development strategies?

EYA4's identification as a breast cancer oncogene with phosphatase activity essential for cancer progression opens opportunities for therapeutic development:

• Target validation approaches:

  • Use domain-specific antibodies to identify critical functional regions

  • Employ phospho-specific antibodies to monitor activity inhibition

  • Develop screening assays for small molecule inhibitors targeting EYA4's serine/threonine phosphatase domain

• Biomarker development:

  • Assess EYA4 expression patterns across cancer types and stages

  • Correlate expression with treatment response and patient outcomes

  • Develop IHC-based diagnostic and prognostic panels incorporating EYA4

• Therapeutic antibody potential:

  • Explore antibodies that specifically inhibit EYA4's phosphatase activity

  • Investigate antibody-drug conjugates targeting EYA4-expressing cancer cells

  • Assess combination approaches with DNA-damaging agents or replication stress inducers

Research indicates that targeting EYA4's serine/threonine phosphatase activity represents a promising strategy for treating breast cancer, potentially limiting metastasis and overcoming chemotherapy resistance caused by endoreplication and genomic rearrangements .

What are the emerging techniques for studying protein-protein interactions involving EYA4?

Understanding EYA4's interactions with other proteins is critical for elucidating its functions in different cellular contexts:

• Advanced co-immunoprecipitation approaches:

  • Tandem affinity purification using tagged EYA4 constructs

  • Proximity-dependent biotinylation (BioID or TurboID) with EYA4 as bait

  • Crosslinking immunoprecipitation for capturing transient interactions

• Microscopy-based interaction studies:

  • Förster resonance energy transfer (FRET) with fluorophore-conjugated antibodies

  • Proximity ligation assay (PLA) to visualize protein interactions in situ

  • Super-resolution microscopy for nanoscale localization of interaction complexes

• Mass spectrometry applications:

  • Immunoprecipitation followed by mass spectrometry (IP-MS)

  • Cross-linking mass spectrometry (XL-MS) for structural insights

  • Targeted proteomics to quantify specific interaction partners

These emerging techniques, combined with high-quality EYA4 antibodies, will advance our understanding of EYA4's molecular functions and potentially identify new therapeutic targets within its interaction network.