Phospho-EPHA7 (Tyr791) Antibody

What is the specificity of Phospho-EPHA7 (Tyr791) antibody and what epitope does it recognize?

The Phospho-EPHA7 (Tyr791) antibody specifically detects endogenous levels of EPHA7 protein only when phosphorylated at tyrosine 791. The antibody is typically generated using synthesized phospho-peptides around the phosphorylation site of human EPHA7 (phospho Tyr791), with the specific epitope sequence being A-V-Y(p)-T-T . The specificity is ensured through affinity purification techniques where non-phospho specific antibodies are removed by chromatography using non-phosphopeptide . This high specificity makes it valuable for distinguishing between phosphorylated and non-phosphorylated forms of EPHA7 in research applications.

What are the validated applications for Phospho-EPHA7 (Tyr791) antibody in research?

Phospho-EPHA7 (Tyr791) antibody has been validated for several research applications:

The antibody has been primarily tested in Western Blot applications with JurKat cells and has demonstrated consistent detection of phosphorylated EPHA7 . While some product descriptions mention potential use in other applications, researchers should conduct preliminary validation in their specific experimental contexts before proceeding with comprehensive studies.

What species reactivity has been confirmed for Phospho-EPHA7 (Tyr791) antibody?

The Phospho-EPHA7 (Tyr791) antibody shows reactivity across multiple species:

This cross-species reactivity makes the antibody valuable for comparative studies across different model organisms . The conservation of the tyrosine 791 phosphorylation site across these species suggests the functional importance of this post-translational modification in EPHA7 signaling.

What are the optimal sample preparation methods for detecting phosphorylated EPHA7 in Western blot applications?

For optimal detection of phosphorylated EPHA7 in Western blot applications:

• Lysis Buffer Composition: Use RIPA buffer supplemented with phosphatase inhibitors (including sodium orthovanadate, sodium fluoride, and β-glycerophosphate) to preserve phosphorylation status.

• Cell Density Considerations: As shown in research findings, the phosphorylation of EPHA7 receptor is regulated by cell density . Sample preparation should maintain consistent cell densities across experimental conditions to avoid misinterpretation of results.

• Protein Loading: Optimal protein loading is typically 20-40μg of total protein per lane.

• Blocking Conditions: Use 5% BSA in TBST rather than milk, as milk contains phosphatases that can reduce phospho-epitope detection.

• Validation Controls: Include samples treated with ephrinA5-Fc as positive controls, as this ligand enhances EPHA7 phosphorylation . Consider including samples with Y791F mutation as negative controls.

Studies have demonstrated that these methodological considerations significantly impact the detection sensitivity and reproducibility of phospho-EPHA7 experiments.

How can researchers effectively validate the specificity of Phospho-EPHA7 (Tyr791) antibody in their experimental system?

To validate the specificity of Phospho-EPHA7 (Tyr791) antibody:

• Peptide Competition Assay: Pre-incubate the antibody with the phospho-peptide immunogen to block specific binding sites. This should eliminate specific signals in Western blot or other applications.

• Genetic Validation: Use cells expressing EPHA7 Y791F mutants as documented in research where tyrosine 791 was identified as critical for EPHA7 receptor phosphorylation . These samples should show absent or significantly reduced signals.

• Phosphatase Treatment: Treat duplicate samples with lambda phosphatase to remove phosphate groups, which should eliminate detection if the antibody is truly phospho-specific.

• Cell Density Experiment: As research has shown that EPHA7 phosphorylation increases with cell density and is further enhanced by ephrinA5 ligand stimulation , compare samples with varying cell densities with and without ligand treatment to confirm expected phosphorylation patterns.

• Cross-validation: Compare results with alternative methods of detecting phosphorylation, such as Phos-tag gels or mass spectrometry.

What considerations should be made when designing co-immunoprecipitation experiments to study EPHA7 interactions using this antibody?

When designing co-immunoprecipitation (co-IP) experiments with Phospho-EPHA7 (Tyr791) antibody:

• Lysis Conditions: Use mild lysis buffers (e.g., NP-40 or Triton X-100 based) with phosphatase inhibitors to maintain protein-protein interactions and phosphorylation status.

• Antibody Amount: Typically 2-5μg of antibody per 500μg-1mg of total protein is recommended for efficient immunoprecipitation.

• Pre-clearing Step: Pre-clear lysates with protein A/G beads to reduce non-specific binding.

• Controls: Include:

  • IgG control to identify non-specific binding

  • Input samples to verify protein expression

  • Phosphatase-treated samples as negative controls

  • Samples with varying cell density as previously shown to affect EPHA7 phosphorylation

• Detection of Interacting Partners: Research has demonstrated that ephrinA5 forms complexes with EPHA7 in LNCaP cell lysates . When investigating such interactions, consider:

  • Whether to use native conditions or crosslinking

  • Whether interacting proteins might mask the Y791 epitope

  • The kinetics of the interaction (transient vs. stable)

Research findings indicate successful co-immunoprecipitation of ephrinA5-EPHA7 complexes can be achieved, supporting the utility of this approach for studying EPHA7 interactions.

How does the phosphorylation of EPHA7 at Tyr791 relate to its tumor suppressive function in prostate cancer?

Research findings have established critical links between EPHA7 Tyr791 phosphorylation and tumor suppression:

• Functional Significance: Studies demonstrate that ligand-dependent EPHA7 signaling resulting in Y791 phosphorylation leads to:

  • Decreased tumor volume

  • Enhanced tumor cell apoptosis in primary tumors

  • Inhibited proliferation, migration, and invasion of prostate cancer cells

• Mechanistic Pathway: The phosphorylation of EPHA7 receptor at Y791 suppresses prostate cancer malignancy through targeting PI3K/Akt signaling pathways . This represents a critical molecular mechanism by which EPHA7 exerts its tumor suppressive functions.

• Ligand Dependency: Importantly, ligand-independent EPHA7 signaling does not produce the same anti-cancer effects, highlighting the importance of ephrinA5-induced phosphorylation at Y791 .

• Clinical Correlation: In prostate cancer tissues, decreased levels of both EPHA7 phosphorylation and ephrinA5 expression have been observed compared to normal tissues. The expression of ephrinA5 positively correlates with the ratio of phosphorylated EPHA7 to total EPHA7 in prostate cancer tissues, suggesting clinical relevance of this phosphorylation event .

This research provides strong evidence that phosphorylation at Y791 is a critical molecular event with significant implications for understanding prostate cancer progression and potential therapeutic approaches.

What is the relationship between cell density, ephrinA5 ligand stimulation, and EPHA7 Tyr791 phosphorylation?

Research has revealed a complex relationship between these factors:

• Cell Density Effects: The level of phosphorylated EPHA7 receptor is upregulated by increasing cell density. This suggests that cell-cell contacts play a role in EPHA7 activation .

• EphrinA5 Expression Correlation: With increasing cell density, ephrinA5 expression levels gradually increase, while EPHA7 expression remains relatively stable . This indicates that ephrinA5 availability may be a limiting factor in EPHA7 activation.

• Exogenous Stimulation Effects: Stimulation with exogenous ephrinA5-Fc further enhances EPHA7 phosphorylation beyond the effects of increased cell density alone . This demonstrates a quantitative relationship between ligand availability and receptor phosphorylation.

• Molecular Mechanism: Experimental evidence from PC-3 cells shows that overexpression of wild-type EPHA7 or juxtamembrane domain mutants results in substantial ligand-independent EPHA7 receptor phosphorylation, which is further enhanced by ephrinA5-Fc treatment . This suggests a two-step activation mechanism.

These findings collectively demonstrate that EPHA7 Tyr791 phosphorylation is dynamically regulated in a cell density-dependent and ligand-dependent manner, with important implications for experimental design and data interpretation.

How do mutations in EPHA7 affect phosphorylation at Tyr791 and downstream signaling pathways?

Research using mutational analysis has provided insights into the structural and functional requirements for EPHA7 phosphorylation:

• Critical Residue Identification: Studies identified Y791 as the primary phosphorylation site of EPHA7. Mutation of this residue (Y791F) in the kinase domain prevents EPHA7 phosphorylation even in the presence of ephrinA5 ligand .

• Domain Requirements: Deletion of the entire cytoplasmic region (ΔCyto mutant) completely abolishes EPHA7 phosphorylation, confirming the necessity of this domain for receptor activation .

• Juxtamembrane Domain Mutations: Interestingly, mutations in the juxtamembrane domain (Y597F/Y608F/Y614F; called DM mutant) do not prevent phosphorylation at Y791, suggesting these residues are not critical for this specific phosphorylation event .

• Functional Consequences:

  • Wild-type EPHA7 overexpression suppresses tumor growth, which requires Y791 phosphorylation

  • The Y791F mutation abolishes EPHA7's ability to inhibit cell proliferation

  • The Y791F mutation prevents EPHA7-mediated inhibition of cell migration and invasion

  • Y791 phosphorylation is necessary for EPHA7's ability to induce prostate carcinoma cell apoptosis

These findings demonstrate that Y791 phosphorylation is both structurally and functionally critical for EPHA7's tumor suppressive activities, with significant implications for understanding oncogenic mechanisms.

What are common technical issues encountered when detecting phosphorylated EPHA7 and how can they be resolved?

Researchers may encounter several challenges when working with Phospho-EPHA7 (Tyr791) antibody:

• Weak or Absent Signal:

  • Cause: Insufficient phosphorylation level, improper sample preparation, or phosphatase activity during preparation

  • Solution: Ensure inclusion of phosphatase inhibitors in all buffers; consider stimulating cells with ephrinA5-Fc to increase phosphorylation levels ; optimize antibody concentration (1:500-1:2000 range recommended for Western blot)

• Multiple Bands:

  • Cause: Cross-reactivity, protein degradation, or alternative phosphorylation sites

  • Solution: Verify using blocking peptides; include protease inhibitors; compare with Y791F mutant controls

• Inconsistent Results:

  • Cause: Variation in cell density affecting phosphorylation levels

  • Solution: Standardize cell densities across experiments; consider the research finding that phosphorylation levels are affected by cell density

• High Background:

  • Cause: Non-specific binding or excessive antibody concentration

  • Solution: Use 5% BSA instead of milk for blocking; optimize antibody dilutions; include proper washing steps

• Discrepancy Between Phospho-Signal and Total Protein:

  • Cause: Biological regulation or technical issues with antibody specificity

  • Solution: Always include detection of total EPHA7 protein; validate with multiple techniques

Research has noted that detection sensitivity can be particularly challenging in cells expressing extremely low levels of EPHA7 protein , suggesting that immunoprecipitation approaches may be necessary in such cases.

How can researchers differentiate between specific EPHA7 Tyr791 phosphorylation and signals from other EphA family members?

Distinguishing specific EPHA7 Tyr791 phosphorylation from other Eph receptors requires careful experimental design:

• Sequence Comparison Analysis: While the Y791 region has sequence similarity across Eph receptors, the antibodies are generated against the specific sequence A-V-Y(p)-T-T . Researchers should:

  • Analyze sequence alignments of Eph family members

  • Identify potential cross-reactive Eph receptors with similar phosphorylation sites

  • Include appropriate controls in experiments

• Validation Approaches:

  • Genetic Validation: Use cells with EPHA7 knockdown or knockout as negative controls

  • Competitive Inhibition: Use blocking peptides specific to phospho-EPHA7 (Tyr791)

  • Cross-Validation: Compare results with alternative detection methods

• Size Discrimination: Different Eph receptors have slightly different molecular weights:

  • EPHA7: ~112 kDa

  • Verify the observed band corresponds to the expected molecular weight

• Interacting Partners: Research has shown that EPHA7 specifically interacts with ephrinA5 . Co-immunoprecipitation experiments can help confirm the identity of the detected receptor.

Given the high homology between Eph family members, these validation steps are critical for ensuring the specificity of observed phosphorylation signals.

What experimental controls should be included when studying the biological effects of EPHA7 Tyr791 phosphorylation?

Based on research methodologies, the following controls are essential:

• Genetic Controls:

  • Wild-type EPHA7 expressing cells

  • Y791F mutant EPHA7 expressing cells (demonstrates phosphorylation-specific effects)

  • ΔCyto mutant EPHA7 expressing cells (lacking the entire cytoplasmic domain)

  • Empty vector control cells

• Stimulation Controls:

  • Unstimulated baseline conditions

  • EphrinA5-Fc stimulated cells (enhances EPHA7 phosphorylation)

  • Dose-response series of ephrinA5 stimulation

  • Time-course of stimulation to capture transient phosphorylation events

• Cell Density Controls:

  • Low density cultures (minimal cell-cell contact)

  • High density cultures (increased cell-cell contact)

  • Mixed population analyses to exclude artifacts

• Technical Controls:

  • Phosphatase-treated samples (negative control)

  • Total EPHA7 protein detection (normalization control)

  • Positive control cell lines (e.g., LNCaP cells which express both endogenous ephrinA5 and EPHA7)

Research has demonstrated that these controls are essential for distinguishing between ligand-dependent and ligand-independent effects, as well as for accurately interpreting the biological significance of EPHA7 phosphorylation.

How does EPHA7 Tyr791 phosphorylation compare across different cancer types, and what are the implications for targeted therapies?

Current research suggests significant variation in EPHA7 phosphorylation patterns across cancer types:

Future research should focus on comprehensive characterization of EPHA7 Y791 phosphorylation across diverse cancer types to identify contexts where targeted activation of this signaling pathway might provide therapeutic benefit.

What methodological advances might improve detection and functional analysis of EPHA7 Tyr791 phosphorylation?

Several emerging methodologies could enhance research on EPHA7 phosphorylation:

• Advanced Detection Methods:

  • Proximity Ligation Assays (PLA): Would allow in situ detection of phosphorylated EPHA7 at the single-molecule level, providing spatial context to phosphorylation events

  • Mass Spectrometry-Based Approaches: Could provide absolute quantification of phosphorylation stoichiometry and identify co-occurring post-translational modifications

  • Phospho-Specific Nanobodies: Could offer improved specificity and access to sterically hindered epitopes

• Live-Cell Imaging Approaches:

  • FRET-Based Biosensors: Would enable real-time monitoring of EPHA7 phosphorylation dynamics in living cells

  • Optogenetic Control of Receptor Clustering: Could provide temporal control over receptor activation independent of ligand availability

• Structural Biology Insights:

  • Cryo-EM Studies: Could reveal conformational changes associated with Y791 phosphorylation

  • Hydrogen-Deuterium Exchange Mass Spectrometry: Could map dynamic structural changes following phosphorylation

• Functional Analysis Tools:

  • CRISPR Base Editing: Would enable precise introduction of Y791F mutations without disrupting protein expression

  • Phosphomimetic Approaches: Could help distinguish between scaffolding and catalytic functions of phosphorylated Y791

These methodological advances would address current limitations in studying this important signaling event, particularly in contexts where endogenous EPHA7 expression is extremely low or where temporal dynamics are critical.

How does EPHA7 Tyr791 phosphorylation integrate with other post-translational modifications to regulate receptor function?

While current research has focused primarily on Y791 phosphorylation, a comprehensive understanding requires consideration of the broader post-translational modification landscape:

• Multiple Phosphorylation Sites:

  • Research has demonstrated the importance of Y791, but EPHA7 contains multiple tyrosine residues that may be phosphorylated .

  • Studies have shown that mutations in the juxtamembrane domain tyrosines (Y597F/Y608F/Y614F) do not prevent Y791 phosphorylation , but these sites may have distinct or cooperative functions.

• Regulatory Interactions:

  • Cross-Regulation: Research has shown interactions between different Eph receptors, such as the finding that EPHA7 TR protein can block EphA2 phosphorylation , suggesting complex regulatory networks.

  • Feedback Mechanisms: The relationship between receptor clustering, phosphorylation, and internalization remains to be fully characterized.

• Beyond Phosphorylation:

  • Ubiquitination: May regulate receptor turnover and availability at the cell surface

  • Glycosylation: Could affect ligand binding and receptor clustering

  • Proteolytic Processing: Research mentions EPHA7 TR (truncated) protein , suggesting regulatory roles for proteolytic events

• Contextual Factors:

  • Cell Density Effects: Research has demonstrated that cell density affects EPHA7 phosphorylation , suggesting membrane organization or mechanical factors may play important roles

  • Membrane Microdomains: Localization to specific membrane domains could affect accessibility to kinases and phosphatases

Future research should take an integrative approach to understanding how these various modifications work together to fine-tune EPHA7 signaling in different cellular contexts.

What are the key considerations for researchers planning to incorporate Phospho-EPHA7 (Tyr791) antibody into their experimental workflow?

Based on the available research and technical information, researchers should consider:

• Experimental Design:

  • Include appropriate controls as outlined in section 4.3

  • Standardize cell densities across experiments due to the demonstrated effect on phosphorylation levels

  • Consider the established ligand-dependency of phosphorylation

• Technical Implementation:

  • Follow optimal sample preparation methods described in section 2.1

  • Use recommended dilutions for specific applications (WB: 1:500-1:2000, ELISA: 1:10000)

  • Store the antibody according to manufacturer recommendations (typically at -20°C in small aliquots to prevent freeze-thaw cycles)

• Data Interpretation:

  • Consider the biological context of EPHA7 phosphorylation, particularly its established role in tumor suppression

  • Assess both phosphorylated and total EPHA7 levels

  • Interpret phosphorylation data in the context of ephrinA5 expression levels

• Validation Approaches:

  • Verify specificity through methods described in section 2.2

  • Cross-validate findings with complementary approaches when possible