EYA2 Antibody

What is EYA2 and what are its main cellular functions?

EYA2 (Eyes Absent Homolog 2) is a member of the EYA family and belongs to the HAD-like hydrolase superfamily. It functions as a protein phosphatase and transcriptional coactivator that interacts with SIX1. EYA2 plays critical roles in organ development, particularly eye development, and is involved in cellular processes including DNA damage response and cell cycle regulation . The protein exhibits both tyrosine phosphatase activity and threonine/serine phosphatase activities that regulate multiple cellular phenotypes associated with tumor progression . EYA2 has three isoforms produced by alternative splicing with molecular weights of approximately 59 kDa, 57 kDa, and 50 kDa, though it is commonly observed at 56 kDa and 70 kDa in Western blot applications .

What types of EYA2 antibodies are available for research applications?

Several validated polyclonal antibodies targeting EYA2 are commercially available, including:

These antibodies have been raised against different EYA2 immunogens and validated for various applications, with each having specific advantages depending on experimental needs .

What are the recommended dilutions and protocols for different applications?

Optimal dilutions vary by application and specific antibody:

For IHC applications, antigen retrieval is recommended using TE buffer at pH 9.0, with citrate buffer pH 6.0 as an alternative option. It is essential to optimize conditions for each experimental system to obtain reliable results .

Why do researchers observe different molecular weights for EYA2 in Western blot analyses?

Though EYA2 has a calculated molecular weight of 64 kDa, researchers commonly observe bands at approximately 56 kDa and 70 kDa in Western blot applications . This discrepancy results from:

• Alternative splicing producing three isoforms (59 kDa, 57 kDa, and 50 kDa)

• Post-translational modifications affecting protein migration

• Cell type-specific processing of the protein

For instance, in neuroblastoma cells, EYA2 appears as two distinct bands at 70 kDa and 56 kDa . When validating antibody specificity, researchers should consider these variations and potentially include positive control samples with known EYA2 expression, such as HeLa cells or Y79 cells, which have been confirmed to express detectable levels of EYA2 .

How does EYA2 expression correlate with breast cancer characteristics and progression?

EYA2 expression shows significant correlations with several clinicopathological features in breast cancer:

Functionally, overexpression of EYA2 enhances proliferation of breast cancer cells as demonstrated through colony-forming assays and EdU experiments, with corresponding increases in proliferation markers at the protein level .

What role does EYA2 play in pancreatic cancer and how can it be studied experimentally?

Research has revealed that EYA2 functions as a tumor suppressor in pancreatic cancer:

• EYA2 is epigenetically silenced in pancreatic adenocarcinomas through both DNA methylation and chromatin modifications

• Experimental restoration of EYA2 expression in pancreatic cancer cell lines results in:

  • Decreased cell proliferation in vitro (4.4-fold reduction in Panc2.5 cells and approximately 2.7-fold reduction in Panc3.014 cells)

  • Reduced tumor growth in vivo (7-fold decrease in subcutaneous models and 3-fold decrease in orthotopic models)

  • Reduced metastatic potential when implanted orthotopically

• Conversely, siRNA-mediated knockdown of EYA2 in Panc215 cells, which naturally express EYA2, resulted in a significant increase in cell proliferation

To study EYA2 function in pancreatic cancer, researchers have employed stable transfection systems with pcDNA6.2/cLumio-DEST containing the EYA2 transcript, with verification of expression by RT-PCR and immunocytofluorescence. The perinuclear localization of EYA2 protein can be visualized through these methods .

How do mutations in specific domains of EYA2 affect its biological functions?

Structure-function analyses of EYA2 have identified critical domains and residues that determine its activity:

• Interaction with the homeodomain transcription factor SIX1 is essential for EYA2's biological functions. Mutants lacking this interaction capability (P510R and A526R) fail to confer aberrant self-renewal capacity on hematopoietic stem/progenitor cells in leukemia models

• The tyrosine phosphatase activity, dependent on residue D268, appears less critical for certain functions. A D268N mutation that reduces enzymatic activity still maintains the ability to immortalize hematopoietic stem cells

• Interaction with SIX1 is functionally relevant, as demonstrated by experiments where a dominant-negative SIX1 mutant lacking its homeodomain (SIX1 ΔHD) suppressed the clonogenicity of EYA2-immortalized cells more effectively than wild-type SIX1

These findings demonstrate that protein-protein interactions, particularly with SIX family transcription factors, are often more critical for EYA2's oncogenic potential than its intrinsic phosphatase activity.

What is known about EYA2's role in DNA damage response pathways?

EYA2 has emerged as an important regulator of DNA damage response:

• Research in regenerative animal models indicates that EYA2 is involved in regulating H2AX phosphorylation, a key event in DNA damage signaling

• Immunoprecipitation experiments have confirmed direct interaction between EYA2 and the phosphorylated form of H2AX (pS139/Y142-H2AX)

• EYA2 appears to be expressed by most cells in wound epidermis and blastema during regeneration, suggesting its importance in regenerative processes

This dual function in transcriptional regulation and DNA damage response makes EYA2 a unique factor connecting gene expression programs with genome integrity maintenance.

What are important considerations when selecting antibodies for chromatin immunoprecipitation (ChIP) studies of EYA2?

Researchers should be aware of limitations when planning ChIP experiments with EYA2 antibodies:

• Previous attempts to perform ChIP analysis to identify EYA2 binding sites reported that available commercial antibodies did not effectively enrich for putative EYA2 targets

• For successful ChIP applications, researchers should:

  • Perform preliminary validation using known EYA2 targets

  • Consider epitope tagging approaches (e.g., FLAG-tagged EYA2) that enable use of well-characterized tag antibodies

  • Include appropriate negative controls (IgG) and positive controls (known EYA2-regulated genes)

  • Validate results through multiple techniques, such as combining ChIP with reporter assays

When studying EYA2's transcriptional activities, alternative approaches like RNA-seq following EYA2 overexpression or knockdown may provide more reliable results for identifying EYA2-regulated genes.

How should researchers address subcellular localization studies of EYA2?

EYA2 can localize to both nuclear and cytoplasmic compartments, which has important implications for its various functions:

• In immunofluorescence studies, researchers have observed EYA2 in both the nucleus and cytoplasm

• For accurate localization studies:

  • Include proper fractionation controls when performing subcellular fractionation (e.g., HDAC1 for nuclear fraction, β-Tubulin for cytoplasmic fraction)

  • When performing immunofluorescence, counterstain with DAPI to clearly define nuclear boundaries

  • Consider context-specific variations, as EYA2 localization may change depending on cell type, cell cycle stage, or stress conditions

• When studying EYA2 in cancer cells that have undergone epithelial-mesenchymal transition, additional markers should be assessed alongside EYA2, including:

  • E-cadherin (epithelial marker)

  • Vimentin (mesenchymal marker)

  • Cytokeratin 18 (epithelial marker)

  • Fibronectin (mesenchymal marker)

These considerations ensure accurate interpretation of EYA2's subcellular distribution and its relationship to cellular phenotypes.