EPHA8 Antibody, HRP conjugated

What is EPHA8 and what are its primary functions in cellular signaling?

EPHA8 (also known as EEK, EK3, and HEK3) belongs to the Eph receptor family of receptor tyrosine kinases that play critical roles in cell-cell communication. The receptor consists of an extracellular region with a globular domain, a cysteine-rich domain, and two fibronectin type III domains, followed by a transmembrane region and cytoplasmic portion . The cytoplasmic region contains a juxtamembrane motif with tyrosine residues serving as autophosphorylation sites, a kinase domain, and a conserved sterile alpha motif (SAM) .

EPHA8 primarily signals through the PI3K and AKT pathways. When activated by ephrin ligands (particularly ephrin-A5, ephrin-A3, and ephrin-A2), EPHA8 undergoes autophosphorylation at key tyrosine residues and triggers downstream signaling cascades . Studies demonstrate that EPHA8 regulates several cellular processes including:

• Enhancement of cell adhesion to fibronectin via α5β1 integrin activation

• Promotion of cell migration through PI3K signaling

• Inhibition of apoptosis via AKT phosphorylation

• Modulation of cytoskeletal reorganization

To investigate EPHA8 signaling experimentally, researchers typically employ:

• Receptor activation using preclustered ephrin-A ligands (ephrin-A5-Fc aggregated with anti-human Fc for 1 hour at 4°C)

• Analysis of downstream pathway components through phospho-specific antibodies

• Time-course experiments (typically 20-30 minutes at 37°C for acute activation)

How does EPHA8 contribute to cancer progression, particularly in breast cancer?

Research has revealed that EPHA8 plays a significant role in breast cancer progression through multiple mechanisms. Studies demonstrate that EPHA8 expression is upregulated in breast cancer tissue compared to paracancerous and benign breast tissue, with high expression significantly associated with tumor size and TNM stage .

EPHA8's oncogenic mechanisms in breast cancer include:

Experimental approaches to study EPHA8 in cancer include:

• Immunohistochemistry on tissue microarrays (with positive staining observed in cytoplasm and membrane of cancer cells)

• Stable knockdown cell lines using shRNA constructs (shRNA-3 demonstrated highest efficiency in silencing EPHA8 expression)

• Functional assays including CCK-8 for proliferation, flow cytometry for apoptosis analysis, and Transwell/wound healing assays for migration and invasion

Importantly, multivariate Cox regression analysis identified that EPHA8 is an independent predictive factor of poor outcome in patients with breast cancer .

What are the optimal sample preparation methods for detecting EPHA8 using HRP-conjugated antibodies?

Optimal sample preparation for EPHA8 detection using HRP-conjugated antibodies varies by application but should follow these methodological guidelines:

For cell lysates:

• Use high phosphate PBS buffer (100 mM phosphate, 150 mM NaCl, pH 7.6)

• Supplement with protease and phosphatase inhibitors to preserve protein integrity

• For membrane proteins, include 0.1-1% non-ionic detergents (NP-40 or Triton X-100)

For Western blotting:

• Denature proteins in loading buffer containing 2% SDS and 5% β-mercaptoethanol

• Use 8-10% SDS-PAGE gels due to EPHA8's large molecular weight (~111 kDa)

• Employ wet transfer methods with PVDF membranes for efficient transfer of large proteins

• Block with 5% non-fat dry milk in TBST for general detection or 5% BSA for phospho-specific detection

• Dilute HRP-conjugated EPHA8 antibodies to 1:500-1:1000 in blocking buffer

For immunohistochemistry/immunofluorescence:

• Optimize fixation protocols (4% paraformaldehyde for cells, formalin for tissues)

• Perform antigen retrieval (heat-induced in citrate buffer pH 6.0 or EDTA buffer pH 9.0)

• Use HRP-conjugated antibodies directly or with appropriate secondary antibodies

• Include positive controls (breast cancer tissues or MCF-7 cells)

For subcellular compartment analysis:

• Use differential centrifugation with specialized extraction buffers

• Analyze membrane and cytoplasmic fractions separately, as membranous and cytoplasmic EPHA8 may have different prognostic implications in breast cancer

How should EPHA8 antibodies be stored to maintain optimal reactivity?

Proper storage of EPHA8 HRP-conjugated antibodies is critical for maintaining reactivity and specificity. Follow these evidence-based storage protocols:

Long-term storage (up to 12 months):

• Store at -20°C in single-use aliquots

• For extended storage (24 months), dilute with up to 50% glycerol before freezing at -2°C to -8°C

• Avoid repeated freeze-thaw cycles which compromise both antibody binding and enzyme activity

Short-term storage (up to 1 month):

• Store at 4°C for frequent use

Physical protection:

• Keep in light-protected vials or cover with aluminum foil to prevent HRP degradation

• Ensure vials are tightly sealed to prevent evaporation

Buffer composition:

• High phosphate PBS (100 mM phosphate, 150 mM NaCl, pH 7.6)

• 0.02% sodium azide as preservative

• 50% glycerol to prevent freeze damage

Usage recommendations:

• Allow antibody to reach room temperature before opening

• Briefly centrifuge before opening to collect solution at the bottom

• Return to appropriate storage conditions immediately after use

Shelf-life monitoring:

• Document date of receipt and track 12-month expiration period

• Periodically validate reactivity using positive control samples

What controls are recommended when using EPHA8 HRP-conjugated antibodies in Western blot analysis?

Implementing comprehensive controls is essential when using EPHA8 HRP-conjugated antibodies in Western blot analysis:

Positive and negative cell line controls:

• Positive: MCF-7 breast cancer cells (high EPHA8 expression)

• Negative: HS-578T breast cancer cells (low EPHA8 expression)

• Normal control: MCF-10A breast epithelial cells

Antibody specificity controls:

• Peptide competition: Pre-incubate antibody with synthetic blocking peptide corresponding to the EPHA8 epitope to demonstrate signal elimination

• Isotype control: Use non-specific IgG from the same host species

• Secondary antibody-only control: Omit primary antibody to identify non-specific binding

Phospho-specific controls (for phospho-EPHA8 detection):

• Lambda phosphatase treatment of lysates to remove phosphate groups

• Parallel blots with antibodies recognizing total EPHA8 for normalization

• Stimulation controls using ephrin ligands (particularly preclustered ephrin-A5-Fc)

Knockdown/knockout validation:

• shRNA knockdown (with shRNA-3 showing highest efficiency)

• CRISPR-Cas9 knockout models to confirm antibody specificity

Technical controls:

• Loading controls (β-actin, GAPDH) on the same blot after stripping or on parallel blots

• Molecular weight markers to confirm band at expected size (~111 kDa)

• Signal linearity verification using serial dilutions of lysate

Example protocol for ephrin stimulation control:

• Aggregate ephrin-A5-Fc using anti-human Fc antibodies (1:10 ratio) for 1 hour at 4°C

• Stimulate cells with preclustered ephrin-A5-Fc for 20-30 minutes at 37°C

• Immediately lyse cells in buffer containing phosphatase inhibitors

• Process for Western blot analysis with HRP-conjugated EPHA8 antibodies

How can researchers effectively knockdown EPHA8 expression to study its function in cancer cell lines?

Effective EPHA8 knockdown requires a systematic approach:

1. Cell model selection:

• Choose models with high endogenous EPHA8 expression (e.g., MCF-7 breast cancer cells)

• Verify baseline expression via Western blot and qRT-PCR before manipulation

2. shRNA-mediated stable knockdown:

• Design multiple shRNA sequences targeting different regions of EPHA8 mRNA

• Clone into appropriate vectors (e.g., pGreenPuro vectors as used in published studies)

• Transfect or transduce target cells followed by selection with puromycin

• Evaluate multiple clones to identify those with highest knockdown efficiency (e.g., shRNA-3)

3. Validation of knockdown efficiency:

• mRNA level: qRT-PCR with EPHA8-specific primers

• Protein level: Western blot using anti-EPHA8 antibodies

• Target 70-90% reduction in expression for functional studies

4. Functional validation:

• Analyze key downstream effectors (phospho-AKT, Bcl-2, p53, Caspase-3, and Bax)

• Conduct phenotypic assays:

  • Proliferation: CCK-8 assay

  • Apoptosis: Annexin V-FITC/PI staining and flow cytometry

  • Invasion: Matrigel Transwell assay

  • Migration: Wound healing assay

5. Rescue experiments:

• Re-express shRNA-resistant EPHA8 variants to confirm phenotype specificity

• Include both wild-type and kinase-inactive (K666R) constructs to determine kinase-dependency

6. Additional approaches for temporally controlled studies:

• Doxycycline-inducible systems using pTRE-EPHA8 and pTet-On constructs

• Selection of positive clones based on EPHA8 expression in the presence of doxycycline (2 μg/ml)

What are the considerations for designing experiments to study EPHA8-mediated cell migration?

Designing robust experiments to study EPHA8-mediated cell migration requires careful attention to multiple factors:

Cell model considerations:

• Characterize EPHA8 expression across multiple cell lines (e.g., MCF-7, HS-578T)

• Consider both loss-of-function (shRNA in high expressors) and gain-of-function (overexpression in low expressors) approaches

• Use inducible expression systems (doxycycline-regulated) to control for adaptation effects

Migration assay selection:

• Wound healing assay: For collective cell migration

  • Create uniform scratches using pipette tips or specialized tools

  • Use serum-reduced media to minimize proliferation effects

  • Capture images at consistent timepoints (0, 24, 48h)

  • Quantify wound closure using image analysis software

• Transwell migration assay: For individual cell chemotactic responses

  • Optimize cell number and incubation time

  • Include appropriate chemoattractants in lower chamber

  • Quantify migrated cells by staining and counting multiple fields

EPHA8 activation strategies:

• Treatment with preclustered ephrin-A5-Fc (aggregated using anti-human Fc)

• PI-PLC treatment to eliminate GPI-linked ephrin-A ligands that might cause autocrine activation

Extracellular matrix considerations:

• Fibronectin coating is particularly relevant as EPHA8 enhances cell adhesion to fibronectin via α5β1 integrin

• Compare migration on different substrates (collagen, laminin, uncoated)

Mechanistic dissection:

• Use domain-specific mutants:

  • Kinase-dead (K666R)

  • Juxtamembrane deletion (EPHA8-ΔJM)

  • SAM domain deletion (EPHA8-ΔSAM)

• Apply pathway inhibitors (PI3K, AKT) to identify signaling dependencies

Visualization approaches:

• Live-cell imaging with EPHA8-EGFP fusion proteins

• Time-lapse microscopy to track cell movement parameters (velocity, directionality)

How can researchers investigate the interaction between EPHA8 and the PI3K/AKT signaling pathway?

Investigating EPHA8's interaction with the PI3K/AKT pathway requires a multi-faceted approach:

1. Physical interaction analysis:

• Co-immunoprecipitation:

  • Immunoprecipitate HA-tagged EPHA8 using anti-HA antibodies

  • Probe for PI3K subunits (particularly p110γ) by Western blot

  • Include stringent washing conditions to confirm specific interactions

  • Compare interactions before and after ephrin-A5 stimulation

• Proximity ligation assay:

  • Visualize protein-protein interactions in situ

  • Use antibodies against EPHA8 and PI3K/AKT components

2. Activation dynamics:

• Stimulate cells with preclustered ephrin-A5-Fc (20-30 minutes at 37°C)

• Assess phosphorylation status:

  • EPHA8 phosphorylation (pY615, pY838, pY839)

  • AKT phosphorylation (pSer473)

  • Western blot with phospho-specific antibodies

3. Genetic approaches:

• EPHA8 variants to identify critical domains:

  • Kinase-dead mutants (K666R)

  • Juxtamembrane domain deletions (EPHA8-ΔJM)

  • SAM domain deletions (EPHA8-ΔSAM)

• PI3K variant co-expression (p110γ-wild-type, p110γ-K833R)

4. Pharmacological interventions:

• PI3K inhibitors (wortmannin, LY294002)

• AKT inhibitors (MK-2206)

• Assess impact on EPHA8-dependent phenotypes

5. Functional readouts:

• PI3K lipid kinase activity (PIP3 production)

• AKT-regulated proteins:

  • Anti-apoptotic: Bcl-2

  • Pro-apoptotic: p53, Caspase-3, Bax

6. Cancer-specific studies:

• Chemoresistance: EPHA8 knockdown increases breast cancer cell sensitivity to paclitaxel through modulation of PI3K/AKT pathway

• Cell survival: Measure apoptosis rates following pathway manipulation

• Analyze correlation between EPHA8 expression and AKT pathway activation in patient tumor samples

How should researchers interpret subcellular localization patterns of EPHA8 in cancer tissues?

Interpreting EPHA8 subcellular localization is critical for understanding its function in cancer:

Membranous versus cytoplasmic expression:

• In breast cancer, an inverse correlation exists between membranous and cytoplasmic EPHA8 expression

• Membranous EPHA8 predicts longer breast cancer survival in both univariate and multivariate analysis

• Cytoplasmic EPHA8 indicates shorter breast cancer survival in univariate analysis

Methodological considerations for accurate interpretation:

• Use high-resolution imaging techniques (confocal microscopy) for precise localization

• Employ membrane markers (e.g., sodium-potassium ATPase) for co-localization studies

• Develop standardized scoring systems to quantify membrane versus cytoplasmic staining

• Consider categorical variables (high/low expression) based on established cutoffs (e.g., staining score ≥140)

Biological significance:

• Membrane-localized EPHA8 likely represents the receptor in its canonical signaling mode

• Cytoplasmic accumulation may indicate:

  • Receptor internalization following ligand binding

  • Mislocalization due to cancer-associated defects in trafficking

  • Non-canonical signaling mechanisms

Clinical correlations:

Verification approaches:

• Subcellular fractionation followed by Western blotting

• Immunofluorescence with z-stack imaging

• Electron microscopy for ultrastructural localization

What factors might affect the detection sensitivity of EPHA8 using HRP-conjugated antibodies?

Multiple factors can impact EPHA8 detection sensitivity with HRP-conjugated antibodies:

Antibody characteristics:

• Epitope specificity: Antibodies targeting the middle region of EPHA8 may provide optimal sensitivity

• Clonality: Monoclonal antibodies offer consistency; polyclonal antibodies provide signal amplification

• Conjugation efficiency: The HRP:antibody ratio affects signal-to-noise ratio

Sample preparation factors:

• Protein extraction methods: Different buffers yield varying efficiency for membrane protein solubilization

• Fixation techniques: Can affect epitope accessibility in immunohistochemistry

• Storage conditions: Repeated freeze-thaw cycles reduce antigen integrity

Technical considerations for Western blot:

• Transfer efficiency: EPHA8's large molecular weight (~111 kDa) requires optimized transfer conditions

• Blocking reagents: 5% milk may be optimal for general detection; BSA for phospho-specific detection

• Substrate selection: Enhanced chemiluminescence versus chromogenic detection systems

Biological variables:

• Expression levels: Vary dramatically across cell types (high in MCF-7, low in HS-578T)

• Post-translational modifications: Phosphorylation at Y615, Y838, or Y839 may mask certain epitopes

• Protein-protein interactions: Binding to ligands or downstream effectors can obscure antibody recognition sites

Optimization strategies:

• Antibody titration: Test dilution series (typically 1:500-1:1000 for Western blot)

• Multiple antibody comparison: Test antibodies targeting different EPHA8 epitopes

• Positive controls: Include MCF-7 lysates as high-expressing control

• Signal enhancement: Consider tyramide signal amplification for low-abundance detection

How should researchers interpret contradictory results regarding EPHA8 expression across different studies?

Interpreting contradictory EPHA8 expression data requires systematic analysis:

Methodological considerations:

• Antibody selection: Different antibodies target distinct epitopes (N-terminal, middle region, C-terminal)

• Detection methods: IHC, Western blot, and qRT-PCR may yield different results due to methodological differences

• Scoring systems: "High" versus "low" expression categories depend on cutoff values (e.g., staining score ≥140)

• Sample preparation: Fixation methods, antigen retrieval protocols affect epitope availability

Biological factors:

• Cancer subtypes: Analyze expression patterns within molecular subtypes rather than entire cancer types

• Tumor heterogeneity: Sampling different regions may yield variable results

• Disease stage: Expression may change during progression (correlate with TNM staging)

• Treatment effects: Prior therapy may alter EPHA8 expression patterns

Resolution approaches:

• Use multiple detection methods (IHC, Western blot, qRT-PCR) on the same samples

• Employ clearly defined scoring criteria with continuous rather than binary expression data

• Conduct multivariate analyses adjusting for clinicopathological variables

• Validate findings in independent cohorts

• Analyze public databases (TCGA, GEO) stratifying by molecular subtypes

Example case study:
Breast cancer studies showed that high EPHA8 expression correlates with poor prognosis , but with important nuance - membranous versus cytoplasmic localization had opposite prognostic implications . This apparent contradiction was resolved by analyzing subcellular distribution patterns rather than total expression.

What are the latest approaches for investigating EPHA8's role in chemoresistance in breast cancer?

Recent research has revealed EPHA8's significant role in chemoresistance, particularly in breast cancer, with innovative approaches being employed:

Genetic manipulation techniques:

• CRISPR-Cas9 knockout of EPHA8 in breast cancer cell lines

• Inducible shRNA systems allowing temporal control of EPHA8 expression

• Rescue experiments with modified EPHA8 constructs to identify critical domains for chemoresistance

Mechanistic investigations:

• Analysis of EPHA8's impact on apoptotic pathway proteins:

  • Anti-apoptotic: Bcl-2 ↑ with EPHA8 expression

  • Pro-apoptotic: p53, Caspase-3, Bax ↓ with EPHA8 expression

• PI3K/AKT pathway activation assessment following chemotherapy exposure

• Combined inhibition of EPHA8 and treatment with paclitaxel

Advanced culture systems:

• 3D tumor spheroids to better recapitulate tumor architecture

• Patient-derived organoids for personalized chemosensitivity testing

• Co-culture systems to evaluate stromal influence on EPHA8-mediated chemoresistance

Translational approaches:

• Correlation between tumor EPHA8 expression and treatment response in patient cohorts

• Analysis of EPHA8 subcellular localization (membranous vs. cytoplasmic) as predictor of chemotherapy response

• Development of EPHA8 inhibitors as chemosensitizing agents

Key experimental protocol:

• Establish stable EPHA8 knockdown in breast cancer cell lines using shRNA (shRNA-3 shown most effective)

• Treat cells with increasing concentrations of paclitaxel (or other chemotherapeutics)

• Assess:

  • Cell viability (CCK-8 assay)

  • Apoptosis (Annexin V-FITC/PI staining)

  • Invasion/migration capacity (Transwell/wound healing assays)

• Analyze changes in AKT pathway components via Western blot

Findings support: Knockdown of EPHA8 significantly increases the sensitivity of breast cancer cells to paclitaxel through inhibition of PI3K/AKT signaling .

What are the challenges in targeting EPHA8 for cancer therapy, and how might these be addressed?

Targeting EPHA8 for cancer therapy presents significant challenges requiring innovative research approaches:

Context-dependent functions:

• EPHA8 exhibits varying roles across cancer types and molecular subtypes

• Membranous versus cytoplasmic localization may have opposing prognostic implications

• Solution: Comprehensive characterization across diverse cancer models and analysis of subcellular localization patterns

Selectivity challenges:

• High homology within the Eph receptor family complicates selective targeting

• Cross-reactivity with other EphA receptors may cause off-target effects

• Solution: Structure-based drug design focusing on unique EPHA8 features; development of highly specific antibodies

Resistance mechanisms:

• Compensatory upregulation of alternative RTKs or downstream effectors

• Pathway redundancy in PI3K/AKT signaling

• Solution: Combination approaches targeting EPHA8 alongside complementary pathways

Therapeutic strategies under investigation:

• Small molecule inhibitors of EPHA8 kinase activity

• Function-blocking antibodies preventing EPHA8-ephrin interaction

• Antisense oligonucleotides or siRNA targeting EPHA8 expression

• Antibody-drug conjugates for targeted delivery to EPHA8-expressing cells

Biomarker development needs:

• Standardized methods for assessing EPHA8 expression and activation status

• Identification of patient subgroups most likely to benefit from EPHA8-targeted therapy

• Correlation of EPHA8 with other molecular markers for refined patient selection

Promising combination approaches:

• EPHA8 inhibition + paclitaxel: Knockdown of EPHA8 significantly increased paclitaxel sensitivity in breast cancer cells

• EPHA8 inhibition + PI3K/AKT inhibitors: Potential for synergistic effects in blocking complementary nodes in the same pathway

Functional validation recommendations:
Researchers should employ comprehensive functional validation including:

• In vitro: Proliferation, apoptosis, migration/invasion assays

• In vivo: Xenograft models with inducible EPHA8 knockdown/overexpression

• Ex vivo: Patient-derived organoids for personalized therapy response prediction