Phospho-EPHA3/EPHA4/EPHA5 (Y779/833) Antibody

What are EPHA3, EPHA4, and EPHA5 receptors and why is phosphorylation at Y779/Y833 important?

EPHA3, EPHA4, and EPHA5 are receptor tyrosine kinases belonging to the Eph receptor family that bind membrane-bound ephrin ligands. These interactions lead to contact-dependent bidirectional signaling between adjacent cells. The phosphorylation at tyrosine residues Y779 in EPHA3/EPHA4 and Y833 in EPHA5 represents a crucial post-translational modification that regulates receptor activation and downstream signaling pathways .

These receptors play important roles in:

• Cell migration and adhesion

• Axon guidance

• Tissue boundary formation

• Cardiac cell migration and differentiation

• Retinotectal mapping of neurons

Phosphorylation at Y779/Y833 is particularly significant as it creates binding sites for SH2-domain-containing proteins like the Crk adaptor, which subsequently activates small GTPases such as RhoA, mediating cytoskeletal reorganization and cell morphology changes .

What are the validated applications for Phospho-EPHA3/EPHA4/EPHA5 (Y779/Y833) antibodies?

Based on the literature and commercial information, these antibodies have been validated for multiple applications:

For optimal results, each application should be optimized with proper controls. For example, when performing IHC, human brain tissue has been validated as a positive control for phospho-EPHA3/EPHA4/EPHA5 (Y779/Y833) antibody detection .

How specific are these antibodies for the phosphorylated forms versus total protein?

Phospho-specific antibodies for EPHA3/EPHA4/EPHA5 (Y779/Y833) are designed to detect these receptors only when phosphorylated at the specified tyrosine residues. According to validation studies, these antibodies show minimal to no cross-reactivity with the non-phosphorylated forms of the receptors .

For example, the EPHA3/EPHA4/EPHA5 (phospho Y779/Y833) polyclonal antibody "detects endogenous levels of human EPHA3/EPHA4/EPHA5 only when phosphorylated at tyrosine 779/833" . Specificity is typically demonstrated through:

• Peptide competition assays (blocking with phospho-peptide eliminates signal)

• Treatment with phosphatase (eliminates signal)

• Stimulation experiments (increases signal)

• Use of kinase inhibitors (reduces signal)

When studying phosphorylation dynamics, it's recommended to run parallel blots with phospho-specific and total protein antibodies to normalize phosphorylation levels to total protein expression .

What are the recommended protocols for detecting phospho-EPHA3/EPHA4/EPHA5 in Western blotting?

For optimal Western blot detection of phosphorylated EPHA3/EPHA4/EPHA5:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors (e.g., sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Use fresh samples or snap-freeze immediately after collection

  • Maintain samples at 4°C during processing

• Gel electrophoresis:

  • Use 7.5-10% gels (receptors are approximately 110-135 kDa)

  • Load 20-50 μg of total protein per lane

• Transfer and blocking:

  • Transfer to PVDF membrane (preferred over nitrocellulose for phospho-proteins)

  • Block with 5% BSA in TBST (not milk, which contains phosphatases)

• Antibody incubation:

  • Dilute primary antibody 1:1000 in 5% BSA/TBST

  • Incubate overnight at 4°C with gentle agitation

  • Wash thoroughly with TBST (4-5 times, 5 minutes each)

  • Use HRP-conjugated secondary antibody (typically 1:2000-1:5000)

• Detection:

  • Use enhanced chemiluminescence (ECL) detection

  • Expected molecular weight is approximately 130-135 kDa

To validate specificity, consider using phosphatase treatment of duplicate samples as a negative control.

How can I induce phosphorylation of EPHA3/EPHA4/EPHA5 receptors in cell culture experiments?

To study phosphorylation dynamics experimentally:

• Ephrin ligand stimulation:

  • EPHA3 preferentially binds ephrin-A5

  • EPHA4 binds ephrin-A1, -A3, -A4, and -A5

  • EPHA5 preferentially binds ephrin-A5

  • Use pre-clustered soluble ephrin-A ligands (1-2 μg/ml) for 5-30 minutes

• Receptor overexpression:

  • Transiently transfect cells with wild-type EPHA3/EPHA4/EPHA5 expression vectors

  • EphA receptors show constitutive tyrosine phosphorylation when overexpressed in HEK 293 cells

• Co-culture systems:

  • Culture target cells with ephrin-expressing cells to induce endogenous activation

  • Separate cells using fluorescent markers or membrane dyes before analysis

For time-course experiments, stimulation with pre-clustered ephrin-A5 typically shows phosphorylation within 5-10 minutes, peaking at 15-30 minutes .

What controls should be included when using phospho-EPHA3/EPHA4/EPHA5 (Y779/Y833) antibodies?

For rigorous scientific experiments, include these controls:

• Positive controls:

  • Cell lines with known expression of phosphorylated EPHA3/EPHA4/EPHA5 (e.g., A431, HEK-293, BxPC-3 cells)

  • Ephrin-A5 stimulated cells (for inducible phosphorylation)

  • Human brain tissue for IHC applications

• Negative controls:

  • Unstimulated cells (baseline phosphorylation)

  • Phosphatase-treated lysates

  • Kinase inhibitor-treated cells

  • EPHA3/EPHA4/EPHA5 knockout or knockdown cells

• Specificity controls:

  • Peptide competition assay (pre-incubation with phosphopeptide should block signal)

  • Parallel detection with non-phospho-specific antibodies

  • Y779F/Y833F mutants (phosphorylation-deficient)

• Loading controls:

  • Total EPHA3/EPHA4/EPHA5 on parallel blots

  • Standard housekeeping proteins (β-actin, GAPDH, tubulin)

When performing immunohistochemistry, include a blocking peptide control as demonstrated in search result , which shows the specificity of the antibody signal when comparing sections with and without blocking peptide preincubation.

How do Y779/Y833 phosphorylation states differ functionally from other phosphorylation sites on EPHA3/EPHA4/EPHA5?

The EPHA3/EPHA4/EPHA5 receptors contain multiple phosphorylation sites with distinct functional roles:

Research has demonstrated that Y779/Y833 phosphorylation is particularly important for:

• Recruitment of SH2-domain containing adaptors

• Cytoskeletal reorganization through RhoA activation

• Cell migration and adhesion dynamics

In contrast, studies of EPHA3 have shown that while Y596 phosphorylation is essential for kinase activity, Y602 and Y779 are not required for activation but serve as major phosphorylation sites for downstream signaling . This functional specialization indicates that Y779/Y833 may be more involved in effector recruitment rather than intrinsic kinase regulation.

How do cancer-associated mutations affect phosphorylation at Y779/Y833 sites?

Cancer somatic mutations in EPHA receptors can significantly impact phosphorylation status and downstream signaling. Several key findings from the research include:

• Mutations in the EPHA3 kinase domain (R728L, K761N, G766E and D806N) abolish tyrosine phosphorylation, including at Y779 .

• The EPHA3 D678E and R728L mutations show reduced autophosphorylation capacity in in vitro kinase assays .

• The A971P mutation in the SAM domain significantly increases tyrosine phosphorylation of EPHA3, suggesting regulatory mechanisms beyond the kinase domain .

When investigating cancer samples or cell lines:

• Compare phosphorylation levels between wild-type and mutant receptors

• Assess correlations between mutation status and clinical outcomes

• Consider how mutations might alter receptor conformation using structural data from EphA5 ligand binding domain studies

These findings suggest that monitoring Y779/Y833 phosphorylation status could serve as a biomarker for functional receptor activity in cancer studies, particularly in lung cancer where EPHA3 mutations have been linked to disease progression .

What is the relationship between EPHA3/EPHA4/EPHA5 phosphorylation and cytoskeletal dynamics?

Research has revealed intricate connections between EPHA receptor phosphorylation and cytoskeletal regulation:

• Phosphorylated Y779 of EPHA3 serves as a binding site for the Crk adaptor protein, which activates RhoA signaling to regulate actin cytoskeletal dynamics .

• PTP-PEST (PTPN12), a central regulator of actin cytoskeletal dynamics, modulates EPHA3 phosphorylation through:

  • Direct dephosphorylation of the receptor

  • Cytoskeletal remodeling effects that indirectly impact receptor clustering

• Ephrin-A5 stimulation induces caspase-3-mediated cleavage of PTP-PEST, generating an N-terminal fragment that attenuates EPHA3 receptor activation and internalization .

• Isolation of EPHA3 receptor signaling clusters from intact plasma membrane fragments shows recruitment of key cytoskeletal and focal adhesion proteins following ephrin stimulation .

• Pharmacological modulation of actin polymerization affects EPHA3 phosphorylation similarly to PTP-PEST overexpression, suggesting cytoskeletal feedback mechanisms .

This bidirectional relationship indicates that EPHA receptor phosphorylation not only triggers cytoskeletal changes but is also regulated by the cytoskeletal environment, creating a dynamic feedback loop important for cell migration and morphological responses.

What are common challenges when detecting phospho-EPHA3/EPHA4/EPHA5 and how can they be addressed?

Researchers often encounter these challenges when working with phospho-EPHA3/EPHA4/EPHA5 antibodies:

• Low signal intensity:

  • Ensure fresh phosphatase inhibitors in all buffers

  • Optimize stimulation conditions (time, concentration)

  • Try signal enhancement systems

  • Increase antibody concentration or incubation time

  • Use more sensitive detection methods (e.g., ECL Advance)

• High background:

  • Increase blocking time/concentration

  • Use 5% BSA instead of milk for blocking

  • Increase washing steps duration/frequency

  • Titrate antibody to optimal concentration

  • Pre-adsorb secondary antibody if needed

• Multiple bands:

  • Verify expected molecular weight (110-135 kDa)

  • Consider receptor processing/degradation products

  • Check for cross-reactivity with related Eph receptors

  • Look for splice variants (mentioned in some commercial resources)

• Inconsistent results:

  • Standardize cell culture conditions and stimulation protocols

  • Use positive controls with every experiment

  • Maintain consistent lysis and processing times

  • Consider the phosphorylation dynamics (rapid turnover)

• Fixation artifacts in IHC/ICC:

  • Optimize fixation time (over-fixation can mask epitopes)

  • Try antigen retrieval methods (especially for phospho-epitopes)

  • Test different fixatives (formaldehyde vs. methanol)

How can I quantitatively assess changes in EPHA3/EPHA4/EPHA5 phosphorylation?

For quantitative analysis of phosphorylation changes:

• Western blot densitometry:

  • Always normalize phospho-signal to total EPHA protein levels

  • Use linear range of detection (validate with dilution series)

  • Include calibration controls on each blot

  • Analyze with software like ImageJ or specific densitometry tools

• ELISA-based methods:

  • Several antibodies are validated for ELISA applications

  • Consider sandwich ELISA with capture antibody against total protein

  • Use phospho-specific antibody for detection

• Phospho-flow cytometry:

  • Allows single-cell resolution analysis

  • Particularly useful for heterogeneous populations

  • Requires validation of antibody performance in flow conditions

• Mass spectrometry:

  • Most precise method for site-specific quantification

  • Consider phospho-enrichment strategies

  • Label-free or isotope-labeled quantification methods

• Proximity ligation assay:

  • Detects protein-protein interactions dependent on phosphorylation

  • Can be used to assess binding of SH2-domain proteins to phosphorylated Y779/Y833

When reporting results, express as fold-change relative to baseline and include statistical analysis of biological replicates (minimum n=3).

How do I distinguish between EPHA3, EPHA4, and EPHA5 phosphorylation in complex samples?

Distinguishing between these highly homologous receptors requires careful experimental design:

• Receptor-specific approaches:

  • Use immunoprecipitation with receptor-specific antibodies before phospho-detection

  • Consider isoform-specific knockdown to confirm band identity

  • Use recombinant standards of each receptor for size comparison

• Mass spectrometry approaches:

  • Analyze tryptic peptides containing the phosphorylation sites

  • Look for unique sequences flanking the Y779/Y833 sites:

    • EPHA3: EAYTTRGK sequence context

    • EPHA4: DVIKPVKVANAFK sequence context

    • EPHA5: QDVIKEILTIGK sequence context

• Expression pattern analysis:

  • EPHA5 is almost exclusively expressed in the nervous system

  • EPHA3 shows highest expression in placenta

  • EPHA4 is more ubiquitously expressed

• Size discrimination:

  • Run high-resolution gels to separate by slight molecular weight differences

  • EPHA3 (110 kDa), EPHA4 (115 kDa), EPHA5 (specific MW may vary)

When absolute specificity is required, consider using receptor-specific antibodies that do not cross-react, even if they detect the same phosphorylation site across multiple receptors.

How can phospho-EPHA3/EPHA4/EPHA5 antibodies be used to study neurological development and disorders?

These antibodies can provide valuable insights into neurological processes:

• Neural development studies:

  • Track EPHA receptor activation during axon guidance and neural circuit formation

  • Study callosal axon guidance, where EPHA3 plays a critical role

  • Investigate retinotectal mapping of neurons, which involves EPHA/ephrin signaling

• Neuroplasticity research:

  • Monitor phosphorylation changes during synaptic remodeling

  • Investigate activity-dependent receptor activation in neuronal cultures

  • Study receptor dynamics in brain slice preparations

• Neurodegenerative disease investigations:

  • Assess altered phosphorylation patterns in disease models

  • Compare receptor activation in patient-derived tissues

  • Correlate phosphorylation changes with disease progression

• Experimental approaches:

  • Immunohistochemistry of developing brain sections

  • Primary neuronal cultures with activity manipulation

  • Brain organoid models for 3D development studies

  • In vivo models with genetic modifications

The high expression of EPHA5 in cortical neurons, cerebellar Purkinje cells, and pyramidal neurons within the cortex and hippocampus makes it particularly interesting for studying region-specific neuronal populations .

What are the emerging roles of EPHA3/EPHA4/EPHA5 phosphorylation in cancer research?

Phosphorylation of these receptors has significant implications in cancer biology:

• Dysregulated phosphorylation:

  • Somatic mutations in EPHA3 functional domains are linked to lung cancer progression

  • Altered phosphorylation patterns correlate with tumor angiogenesis and progression in gastric and colorectal carcinoma

• Diagnostic applications:

  • Phosphorylation status as potential biomarkers

  • Association with specific cancer subtypes or stages

  • Correlation with treatment resistance

• Therapeutic targeting:

  • Monitoring treatment responses to receptor tyrosine kinase inhibitors

  • Developing phosphorylation-state specific targeting strategies

  • Combining with cytoskeletal-targeting therapies based on PTP-PEST regulation findings

• Research strategies:

  • Phosphoproteomics of patient samples

  • Patient-derived xenograft models

  • Correlation of mutation status with phosphorylation patterns

  • Drug screening platforms monitoring phosphorylation changes

EphA2, a related receptor, has been identified as an oncoprotein and essential survival factor for many melanomas, suggesting similar roles might exist for EPHA3/EPHA4/EPHA5 in other cancer types .

How do computational approaches integrate with phospho-EPHA3/EPHA4/EPHA5 experimental data?

Modern research increasingly combines experimental phosphorylation data with computational approaches:

• Structural modeling:

  • Crystal structure of the EphA5 ligand binding domain reveals unique structural features even in the unbound state

  • Molecular dynamics (MD) simulations provide insights into conformational changes upon phosphorylation

  • Structure-based drug design targeting phosphorylation-dependent conformations

• Signaling pathway analysis:

  • Integration of phosphorylation data into pathway models

  • Prediction of downstream effects based on altered phosphorylation

  • Systems biology approaches to understand network-level consequences

• Machine learning applications:

  • Pattern recognition in phosphorylation datasets across cancer types

  • Prediction of functional outcomes from phosphorylation profiles

  • Integration with other -omics data for comprehensive analysis

• Data visualization tools:

  • Mapping phosphorylation sites to protein structures

  • Network visualization of phospho-dependent interactions

  • Temporal dynamics modeling of phosphorylation cascades