EPS8 Antibody

What is EPS8 protein and what cellular functions does it regulate?

EPS8 (Epidermal Growth Factor Receptor Pathway Substrate 8) is an actin regulatory scaffold protein that plays crucial roles in cellular signaling pathways, particularly in the context of receptor tyrosine kinases (RTKs). The protein exists in two isoforms with molecular masses of 68 kDa and 97 kDa, which are proposed to be alternative splice isoforms or proteolytic products . EPS8 is primarily located in the cytoplasm and at the cell membrane, where it interacts with various signaling molecules to influence actin dynamics and cellular morphology .

The protein consists of an N-terminal phosphotyrosine-binding (PTB) domain, an SH3 domain, and a C-terminal effector domain. Through these domains, EPS8 directs actin regulatory functions, such as capping barbed ends and promoting actin bundling . When investigating EPS8's cellular functions, researchers should consider its dual role in both normal cell signaling and pathological conditions, as dysregulation of EPS8 and associated pathways can lead to aberrant cell behavior and contribute to oncogenesis .

What are the available types of EPS8 antibodies and their specific applications?

Various types of EPS8 antibodies are available for research applications, with different species reactivity and conjugation options:

When selecting an EPS8 antibody, researchers should consider the specific experimental application and required species reactivity. For multi-color flow cytometry or fluorescence microscopy, the conjugated versions offer advantages, while for applications requiring signal amplification, the HRP-conjugated versions may be preferable. For co-immunoprecipitation studies examining EPS8 interactions with partners like FAK or Src, non-conjugated antibodies used with appropriate secondary detection systems typically yield optimal results .

How does EPS8 expression differ between normal and cancer cells?

EPS8 expression is significantly elevated in squamous cell carcinoma (SCC) cells compared to normal keratinocytes. This upregulation has been documented in both murine models and human patient samples .

In a study using the DMBA/TPA model of chemical carcinogenesis, SCC cells (designated SCC 1 and SCC 2) displayed markedly higher EPS8 expression than primary keratinocytes isolated from mouse tails. Furthermore, malignant SCC subclones derived from the SCC 1 cell line (subclones 1-1 and 1-2) expressed substantially higher EPS8 levels than primary keratinocytes .

In human samples, eight out of nine SCC cell lines showed enhanced EPS8 expression compared to normal human keratinocytes (NHKs). The elevated expression primarily resulted from increased transcription, with three out of four mouse and eight out of nine human SCC cell lines showing increased Eps8 mRNA compared to their normal counterparts .

When designing experiments to assess EPS8's role in cancer, researchers should include appropriate normal cell controls and consider examining both mRNA and protein expression levels, as the correlation between transcriptional upregulation and protein abundance provides more comprehensive insights into EPS8's involvement in the cancer phenotype.

How does EPS8 interact with focal adhesion kinase (FAK) in cancer cells, and what are the implications for cell invasion?

EPS8 forms a complex with FAK at focal adhesions in SCC cells, which has significant implications for cancer cell invasion. Co-immunoprecipitation studies using either anti-EPS8 or anti-FAK antibodies have confirmed this interaction in FAK-expressing SCC cells .

The EPS8-FAK interaction occurs specifically at the focal-adhesion-targeting (FAT) domain of FAK, spanning amino acids 981–1053. Peptide array binding analysis identified lysine residues K1001 and K1003 in FAK as critical for binding to EPS8. When these residues were mutated to alanine (K1001A/K1003A), the interaction between FAK and EPS8 was significantly impaired .

The functional significance of this interaction is evident in invasion assays. SCC cells expressing the FAK K1001A/K1003A mutant showed significantly reduced invasion through Matrigel compared to cells expressing wild-type FAK. Similarly, Eps8 knockdown in FAK-expressing SCC cells resulted in more than fivefold inhibition of invasion capacity .

When investigating this interaction, researchers should consider:

• Using both co-immunoprecipitation and colocalization studies to confirm the interaction

• Employing site-directed mutagenesis to study specific binding domains

• Validating functional consequences through invasion assays

• Examining downstream signaling effects that may mediate the invasion phenotype

What role does EPS8 play in Src trafficking and autophagic targeting in FAK-deficient cancer cells?

In FAK-deficient SCC cells, EPS8 participates in a biochemical complex that controls the targeting of active Src to autophagic structures. This represents a cellular mechanism to cope with high levels of active Src when FAK is absent .

Co-immunoprecipitation experiments revealed that EPS8 interacts with phosphorylated Src (p-Src) in both FAK-expressing and FAK-deficient cells. This indicates that the complex between EPS8 and active Src does not depend on the EPS8 interaction with FAK .

These findings suggest that in the absence of proper scaffolding by FAK at focal adhesions, EPS8 helps redirect active Src to autophagosomes. This mechanism likely represents a cellular adaptation to protect against the potentially harmful effects of mislocalized hyperactive Src kinase .

For researchers studying this phenomenon, methodological approaches should include:

• Combined use of genetic knockout/knockdown with immunofluorescence to track protein localization

• Co-immunoprecipitation under various conditions (FAK present/absent)

• Autophagy inhibitors or markers to confirm the autophagic nature of the puncta

• Live-cell imaging to observe the dynamics of this trafficking process

How do the different isoforms of EPS8 (68 kDa and 97 kDa) differ in their expression patterns and functions?

EPS8 exists in two isoforms with molecular masses of 68 kDa and 97 kDa, which are proposed to be alternative splice isoforms or proteolytic products. The expression patterns of these isoforms show tissue and cell-type specificity .

In murine SCC cell lines, both isoforms can be expressed, but their distribution varies:

• The SCC 1 cell line expressed both isoforms

• The SCC 1-2 subclone predominantly expressed the 68-kDa form

• The SCC 1-1 subclone predominantly expressed the 97-kDa form

• The SCC 2 cell line expressed both isoforms to a similar extent

In contrast, human SCC cell lines predominantly expressed the 97-kDa form of EPS8 .

While the functional significance of these different expression patterns remains poorly characterized, most studies refer mainly to the 97-kDa isoform. Both isoforms contain the structural domains (PTB, SH3, and C-terminal effector domains) that enable EPS8 to participate in actin regulation and signaling pathways .

For researchers investigating isoform-specific functions, methodological considerations should include:

• Using antibodies that can distinguish between the isoforms

• Employing isoform-specific siRNA knockdown when possible

• Expressing recombinant versions of each isoform in appropriate model systems

• Designing experiments that can assess potential functional differences in actin regulation, protein interactions, or cellular localization

What are the optimal protocols for detecting EPS8 by Western blotting in different cell lines?

Detecting EPS8 by Western blotting requires optimization based on the cell line and isoform expression pattern. Based on the available data, here is a recommended protocol:

Sample Preparation:

• Lyse cells in RIPA buffer supplemented with protease and phosphatase inhibitors

• Sonicate briefly to shear DNA and reduce sample viscosity

• Centrifuge at 14,000×g for 15 minutes at 4°C to remove debris

• Determine protein concentration using a Bradford or BCA assay

Western Blotting Parameters:

• Protein loading: 20-50 μg of total protein per lane

• Gel percentage: 8% SDS-PAGE (provides optimal separation for 68-97 kDa proteins)

• Transfer: Semi-dry or wet transfer to PVDF membrane

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

• Primary antibody: Human EPS8 Antigen Affinity-purified Polyclonal Antibody (1 μg/mL) or EPS8 Antibody (F-8) at 1:1000 dilution

• Incubation: Overnight at 4°C with gentle rocking

• Secondary antibody: HRP-conjugated Anti-Goat IgG or appropriate secondary based on primary antibody species

Expected Results by Cell Line:

• A431 human epithelial carcinoma: Strong band at approximately 97 kDa

• MCF-7 human breast cancer: Detectable band at approximately 97 kDa

• MDA-MB-468 human breast cancer: Detectable band at approximately 97 kDa

• A549 human lung carcinoma: Detectable band at approximately 97 kDa

For mouse SCC cell lines, researchers should be prepared to detect both the 68 kDa and 97 kDa isoforms, with expression patterns varying by specific cell line as detailed in section 2.3 .

How can researchers effectively use EPS8 antibodies for co-immunoprecipitation studies of protein-protein interactions?

Co-immunoprecipitation (Co-IP) is a valuable technique for studying EPS8 interactions with partners like FAK and Src. Based on published methodologies, here is an effective protocol:

Co-IP Protocol for EPS8 Interactions:

• Cell Lysis:

  • Wash cells twice with ice-cold PBS

  • Lyse in ice-cold IP lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, with protease and phosphatase inhibitors)

  • Incubate on ice for 30 minutes with occasional mixing

  • Centrifuge at 14,000×g for 15 minutes at 4°C

• Pre-clearing (reduces non-specific binding):

  • Incubate lysate with Protein A/G beads for 1 hour at 4°C

  • Remove beads by centrifugation

• Immunoprecipitation:

  • Option A: Incubate 1-2 mg of pre-cleared lysate with 2-5 μg of EPS8 antibody overnight at 4°C

  • Option B: For reverse Co-IP, use antibodies against suspected interaction partners (FAK, Src)

  • Add 30-50 μl of Protein A/G beads and incubate for 2-4 hours at 4°C

  • Wash beads 4-5 times with IP wash buffer (lysis buffer with reduced detergent)

• Elution and Analysis:

  • Elute proteins by boiling beads in 2X Laemmli sample buffer

  • Analyze by SDS-PAGE and Western blotting, probing for both the immunoprecipitated protein and suspected interaction partners

Special Considerations:

• When studying EPS8-FAK interactions, use antibodies against the FAT domain of FAK

• For EPS8-Src interactions, antibodies that specifically recognize phosphorylated Src provide insights into active Src-EPS8 complexes

• Consider crosslinking the antibody to the beads to prevent antibody bands from interfering with detection

• Include appropriate controls: IgG control, input sample (5-10% of lysate), and when possible, samples with knockdown of the target protein

This approach has successfully demonstrated interactions between EPS8 and both FAK and Src in SCC cells, with the interactions confirmed by reciprocal Co-IPs .

What immunofluorescence protocols best visualize EPS8 localization in relation to focal adhesions and autophagic structures?

Visualizing EPS8 in relation to other cellular structures requires optimized immunofluorescence protocols. Based on research findings, here is a recommended approach:

Immunofluorescence Protocol for EPS8 Localization:

• Cell Preparation:

  • Plate cells on fibronectin-coated (10 μg/ml) glass coverslips

  • For focal adhesion studies: Allow cells to adhere for 24 hours

  • For autophagy studies: Consider serum starvation or treatment with autophagy inducers

• Fixation and Permeabilization:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Wash three times with PBS

  • Permeabilize with 0.1% Triton X-100 in PBS for 5 minutes

  • Wash three times with PBS

• Blocking and Antibody Incubation:

  • Block with 5% BSA in PBS for 1 hour at room temperature

  • Incubate with primary antibodies overnight at 4°C:

    • Anti-EPS8 (1:100 dilution)

    • For focal adhesions: Anti-FAK or anti-paxillin (1:100)

    • For Src co-localization: Anti-phospho-Src (1:100)

    • For autophagic structures: Anti-LC3B (1:200)

  • Wash three times with PBS

  • Incubate with appropriate fluorescent secondary antibodies (1:500) for 1 hour at room temperature

  • Wash three times with PBS

• Actin Visualization (optional):

  • Include fluorescently labeled phalloidin (1:200) during secondary antibody incubation

• Mounting and Imaging:

  • Mount coverslips using anti-fade mounting medium with DAPI

  • Image using confocal microscopy with appropriate filter sets

Expected Localization Patterns:

• In FAK-expressing cells: EPS8 colocalizes with FAK at peripheral focal adhesions

• In FAK-deficient cells: EPS8 colocalizes with phospho-Src in intracellular puncta that also contain autophagy markers

• For polarization studies: Visualize the Golgi (using anti-GM130) to assess cell polarization toward a wound

This protocol has successfully demonstrated the differential localization of EPS8 depending on FAK status, revealing its dual roles in focal adhesion function and autophagosomal targeting of active Src .

How can researchers address variability in EPS8 antibody performance across different experimental applications?

Variability in antibody performance is a common challenge in EPS8 research. Here are methodological approaches to address this issue:

Antibody Validation Strategies:

• Western Blot Optimization:

  • Test multiple antibody concentrations (0.1-2 μg/ml range)

  • Evaluate different blocking agents (5% milk vs. 5% BSA)

  • Compare different detection systems (chemiluminescence vs. fluorescence)

  • Include positive controls (cell lines known to express EPS8, e.g., A431 cells)

  • Include negative controls (EPS8 knockdown samples)

• Immunoprecipitation Optimization:

  • Test different lysate concentrations (0.5-2 mg total protein)

  • Vary antibody amounts (1-5 μg per IP)

  • Adjust incubation times (overnight vs. shorter incubations)

  • Modify wash stringency based on background levels

• Immunofluorescence Optimization:

  • Test multiple fixation methods (PFA vs. methanol)

  • Evaluate antigen retrieval techniques if necessary

  • Compare different permeabilization reagents (Triton X-100 vs. saponin)

  • Titrate antibody concentrations (1:50 to 1:500 range)

  • Block with serum from the species of the secondary antibody

• Cross-Application Validation:

  • Confirm results using multiple techniques (e.g., verify IF results with WB)

  • When possible, use antibodies recognizing different epitopes

  • Validate findings using genetic approaches (siRNA, CRISPR-Cas9)

For researchers studying both isoforms, it's important to select antibodies that can detect both the 68 kDa and 97 kDa forms, or to use isoform-specific antibodies when focusing on one particular variant .

What controls should be included when studying EPS8's role in cancer cell invasion and migration?

When investigating EPS8's role in cancer cell invasion and migration, proper controls are essential for robust and reproducible results:

Essential Controls for Invasion and Migration Assays:

• Genetic Controls:

  • Complete EPS8 knockdown (siRNA or shRNA)

  • Partial EPS8 knockdown to assess dose-dependency

  • Rescue experiments with re-expression of siRNA-resistant EPS8

  • Isoform-specific knockdown and rescue

  • Non-targeting siRNA/shRNA controls

• Molecular Pathway Controls:

  • FAK-positive and FAK-negative cells

  • Cells expressing FAK mutants that cannot bind EPS8 (K1001A/K1003A)

  • Src inhibition (e.g., with PP2 or dasatinib)

  • Actin cytoskeleton disruption (e.g., with cytochalasin D)

• Assay-Specific Controls:

  • For wound healing: Proliferation controls (mitomycin C treatment)

  • For Transwell assays: Membrane-only vs. Matrigel-coated

  • For invasion assays: Different extracellular matrix components

  • Cell viability assessments parallel to migration/invasion experiments

• Cell Line Controls:

  • Multiple cell lines with varying EPS8 expression levels

  • Normal vs. cancer cells from the same tissue

  • Cancer cells with different invasive potentials

Quantification and Analysis Recommendations:

• Conduct time-course experiments rather than single endpoints

• Use automated, unbiased image analysis when possible

• Present data as fold-change relative to appropriate controls

• Perform statistical analysis across multiple independent experiments (n≥3)

In published studies, these controls have helped establish that EPS8 is specifically required for FAK-dependent cancer cell invasion, with FAK-deficient cells showing no further suppression of wound closure upon EPS8 knockdown, suggesting their effects are linked within the same pathway .

How can researchers differentiate between the scaffolding and actin-regulatory functions of EPS8 in experimental settings?

EPS8 has dual functions as both a scaffolding protein in signaling complexes and a direct regulator of actin dynamics. Distinguishing between these functions requires specific experimental approaches:

Methodological Approaches to Separate EPS8 Functions:

• Domain-Specific Mutants:

  • Express the SH3 domain alone (disrupts actin regulation but maintains some scaffolding)

  • Express the C-terminal effector domain alone (preserves actin regulatory functions)

  • Create point mutations in the actin-binding regions without affecting scaffold binding sites

  • Use deletion mutants lacking specific functional domains

• Protein Interaction Analysis:

  • Perform proximity ligation assays (PLA) to visualize specific protein interactions in situ

  • Use fluorescence resonance energy transfer (FRET) to detect direct protein interactions

  • Conduct size-exclusion chromatography to isolate different EPS8-containing complexes

  • Employ BioID or proximity-dependent biotin identification to map the EPS8 interactome

• Actin Dynamics Assays:

  • Measure actin polymerization rates in the presence of wild-type or mutant EPS8

  • Visualize actin dynamics using fluorescent actin probes (LifeAct, SiR-Actin)

  • Perform fluorescence recovery after photobleaching (FRAP) on actin structures

  • Use super-resolution microscopy to examine EPS8 localization relative to actin filaments

• Functional Separation Experiments:

  • Disrupt actin dynamics using pharmaceuticals while preserving EPS8 scaffolding

  • Compare phenotypes between actin-binding mutants and signaling-deficient mutants

  • Express competing peptides that specifically block either scaffolding or actin-regulatory functions

  • Perform temporal analysis to determine which function precedes the other

When studying EPS8's interaction with Abi-1 through its SH3 domain, researchers should consider that this interaction releases autoinhibitory binding within EPS8 and promotes actin capping functions. This represents a case where the scaffolding function (binding to Abi-1) directly influences the actin-regulatory function, highlighting the interconnected nature of these roles .

What emerging research directions are most promising for understanding EPS8's role in cancer progression?

Several promising research directions are emerging in the field of EPS8 research, particularly related to cancer progression:

• Therapeutic Targeting Strategies:

  • Development of small molecule inhibitors targeting the EPS8-FAK interaction

  • Peptide-based approaches to disrupt specific protein-protein interactions

  • Exploration of EPS8 as a biomarker for cancer progression or treatment response

  • Investigation of combination therapies targeting both EPS8 and its interaction partners

• Mechanistic Studies:

  • Detailed mapping of the EPS8 interactome in different cancer contexts

  • Investigation of isoform-specific functions and their relevance to cancer progression

  • Examination of post-translational modifications regulating EPS8 functions

  • Studies on EPS8's role in cancer stem cells and tumor heterogeneity

• In Vivo Validation:

  • Generation of conditional EPS8 knockout mouse models

  • Development of patient-derived xenografts with EPS8 manipulation

  • In vivo imaging of EPS8-dependent processes during tumor progression

  • Assessment of EPS8 as a target in immunotherapy approaches

• Clinical Correlations:

  • Comprehensive analysis of EPS8 expression across cancer types and stages

  • Correlation of EPS8 levels with patient outcomes and treatment responses

  • Investigation of EPS8 in therapy resistance mechanisms

  • Exploration of EPS8 as a liquid biopsy biomarker

These research directions build upon the established roles of EPS8 in cancer cell signaling, actin dynamics, and invasion, while expanding into therapeutic applications and deeper mechanistic understanding .