Phospho-ERBB2 (Y1139) Recombinant Monoclonal Antibody

What is the functional significance of ERBB2 phosphorylation at Y1139 versus other phosphorylation sites?

Phosphorylation of ERBB2 at Y1139 has distinct biological significance compared to other phosphorylation sites. Y1139 phosphorylation is predominantly associated with apical ERBB2 in polarized cells, while Y1248 phosphorylation is observed with basolateral ERBB2 . This spatial distinction has significant functional implications:

• Y1139 phosphorylation creates a docking site for the Grb2 adaptor protein

• Y1248 phosphorylation creates binding sites for Shc adaptor proteins

• These different adaptor proteins activate distinct downstream signaling pathways

Research has demonstrated that Y1139 phosphorylation leads to activation of p38 and upregulation of Bcl-2, contributing to cell survival pathways . In contrast, Y1248 phosphorylation primarily couples ERBB2 to the Ras-Raf-MAP kinase signal transduction pathway associated with cell proliferation .

Which experimental applications are recommended for Phospho-ERBB2 (Y1139) Recombinant Monoclonal Antibody?

Phospho-ERBB2 (Y1139) Recombinant Monoclonal Antibody has been validated for multiple experimental applications:

For optimal results in Western blot applications, researchers should use positive controls such as A-431 epithelial carcinoma cells treated with EGF, which enhances Y1139 phosphorylation . The antibody recognizes human ERBB2 protein phosphorylated specifically at the Y1139 residue, with an observed molecular weight of approximately 185 kDa .

How does Phospho-ERBB2 (Y1139) Recombinant Monoclonal Antibody differ from conventional monoclonal antibodies?

Phospho-ERBB2 (Y1139) Recombinant Monoclonal Antibody is synthetically generated in vitro through a precisely controlled process that offers several advantages over conventional antibodies:

• Production Process: The antibody is generated by first extracting ERBB2 antibody genes from B cells isolated from immunoreactive rabbits. These genes are then amplified and cloned into phage vectors, which are subsequently introduced into mammalian cell lines for antibody production .

• Purification Method: The antibody is purified from culture supernatant through affinity chromatography, ensuring high purity and specificity .

• Consistency Advantages: Recombinant production eliminates batch-to-batch variability common in traditional hybridoma-derived antibodies, providing consistent performance across experiments .

• Specificity: The recombinant antibody demonstrates high specificity for the phosphorylated Y1139 epitope with minimal cross-reactivity to other phosphorylation sites or non-phosphorylated ERBB2 .

How should positive and negative controls be designed for experiments using Phospho-ERBB2 (Y1139) antibody?

Appropriate controls are critical for experiments using phospho-specific antibodies:

Positive Controls:

• A-431 human epithelial carcinoma cells treated with EGF (10 ng/mL for 5 minutes)

• SKOV-3 ovarian cancer cells, which naturally express ERBB2 phosphorylated at Y1139

• Synthetic phosphorylated peptide containing the Y1139 sequence (PEYV N)

Negative Controls:

• Untreated cell lines that express ERBB2 but have low basal phosphorylation

• Cell lines treated with ERBB2 kinase inhibitors (e.g., lapatinib)

• Antibody blocking with the immunizing phosphopeptide

• Phosphatase treatment of samples to remove phosphorylation

Validation Strategy:

• Compare phospho-ERBB2 (Y1139) levels before and after EGF stimulation

• Verify specificity using ERBB2 kinase inhibitors to block phosphorylation

• Confirm total ERBB2 expression using a separate pan-ERBB2 antibody

• Use genetic knockdown/knockout models to confirm antibody specificity

What sample preparation techniques maximize detection of Phospho-ERBB2 (Y1139) in Western blot experiments?

Optimal detection of phosphorylated proteins requires careful sample preparation:

• Cell Lysis Buffer Composition:

  • Use RIPA or NP-40 buffer containing:

    • Phosphatase inhibitors (sodium orthovanadate 1mM, sodium fluoride 10mM)

    • Protease inhibitors (PMSF 1mM, protease inhibitor cocktail)

    • EDTA (1mM)

  • Maintain cold temperature (4°C) throughout processing

• Stimulation Protocol:

  • For maximal Y1139 phosphorylation, stimulate cells with:

    • EGF (10 ng/mL for 5 minutes)

    • Pervanadate (1mM for 10 minutes) to inhibit phosphatases

• SDS-PAGE Conditions:

  • Use 7.5% gels for optimal resolution of the 185 kDa ERBB2 protein

  • Include reducing conditions (β-mercaptoethanol)

  • Use freshly prepared samples whenever possible

• Transfer Conditions:

  • Transfer proteins to PVDF membrane (preferred over nitrocellulose)

  • Use wet transfer at low voltage (30V) overnight at 4°C

• Blocking and Antibody Incubation:

  • Block with 5% BSA in TBS-T (not milk, which contains phosphatases)

  • Optimal antibody dilution: 1:100 to 1:500

  • Incubate overnight at 4°C with gentle agitation

How can multiple ERBB2 phosphorylation sites be simultaneously analyzed in experimental systems?

Analyzing multiple phosphorylation sites provides a comprehensive understanding of ERBB2 activation status and downstream signaling:

Methodological Approach:

• Multiplex Western Blotting:

  • Sequential probing with different phospho-specific antibodies

  • Use fluorescent secondary antibodies with distinct emission spectra

  • Strip and reprobe membranes with caution that stripping may reduce signal

• Phospho-Flow Cytometry:

  • Simultaneously measure multiple phosphorylation sites at single-cell level

  • Use different fluorophore-conjugated phospho-specific antibodies

  • Particularly useful for heterogeneous cell populations

• Phospho-Proteomics:

  • Mass spectrometry-based approach for unbiased assessment

  • Can identify all phosphorylation sites simultaneously

  • Sample preparation protocol:

    1. Immunoprecipitate total ERBB2

    2. Digest with trypsin

    3. Enrich phosphopeptides using TiO₂ or IMAC

    4. Analyze by LC-MS/MS

• Proximity Ligation Assay (PLA):

  • Visualize and quantify specific phosphorylation events in situ

  • Combine antibodies against total ERBB2 and different phospho-sites

  • Enables spatial analysis of phosphorylation patterns

Data Integration Strategy:
Create phosphorylation pattern profiles by calculating the relative ratios of different phosphorylation sites (e.g., pY1139:pY1248) to correlate with biological outcomes or therapeutic responses.

How do I interpret differences between total ERBB2 levels and Phospho-ERBB2 (Y1139) signals?

Discrepancies between total ERBB2 and phospho-ERBB2 (Y1139) signals provide important biological insights:

Interpretation Framework:

• High total ERBB2, low pY1139:

  • May indicate inactive receptor status

  • Could suggest inhibition of upstream kinases

  • Potential presence of active phosphatases

  • Possible antagonistic phosphorylation at other sites

• Low total ERBB2, high pY1139:

  • May indicate highly efficient phosphorylation

  • Possible stabilization of phosphorylated form

  • Could reflect selective antibody sensitivity issues

  • Consider rapid receptor turnover after phosphorylation

• Tissue-specific patterns:

  • Studies show that in HER2-positive breast cancers, 62.5% express phosphorylated HER2

  • Even 12.5% of HER2-negative cancers can display phosphorylated HER2

  • Correlation between phosphorylation status and clinical parameters (e.g., younger patient age, negative estrogen receptor status)

Quantification Approach:
Calculate the phosphorylation ratio (pY1139/total ERBB2) to normalize for variations in total protein expression and enable more accurate comparisons between experimental conditions.

What are the most common technical issues with Phospho-ERBB2 (Y1139) antibody experiments and how can they be resolved?

Phospho-specific antibody experiments present several technical challenges:

Validation Strategy:
To confirm antibody specificity:

• Perform peptide competition assays using phosphorylated and non-phosphorylated Y1139 peptides

• Compare results with other phospho-ERBB2 (Y1139) antibodies from different vendors

• Use cells expressing ERBB2 with Y1139F mutation as negative controls

How do experimental conditions affect the detection of Phospho-ERBB2 (Y1139)?

The phosphorylation status of ERBB2 at Y1139 is dynamically regulated and highly sensitive to experimental conditions:

• Cell Density Effects:

  • Confluent epithelial cells show distinct apical vs. basolateral phosphorylation patterns

  • Y1139 phosphorylation is predominantly found in apical ERBB2 in polarized cells

  • Cell-cell contacts can alter receptor dimerization and phosphorylation

• Growth Factor Stimulation:

  • EGF stimulation (10 ng/mL, 5 minutes) enhances Y1139 phosphorylation

  • Different growth factors may induce distinct phosphorylation patterns

  • Kinetics of phosphorylation vary by site (rapid vs. sustained)

• Microenvironment Factors:

  • Matrix components can alter ERBB2 dimerization and phosphorylation

  • Hypoxia affects phosphorylation patterns

  • pH changes influence kinase activity and phosphatase function

• Receptor Localization:

  • Unique phosphorylation patterns exist between apical vs. basolateral ERBB2

  • Apical ERBB2 is primarily phosphorylated at Y1139

  • Basolateral ERBB2 is predominantly phosphorylated at Y1248

Experimental Strategy:
To ensure reproducible phosphorylation detection:

• Standardize cell culture conditions (density, media composition, passage number)

• Control timing precisely during stimulation and lysis steps

• Document and maintain consistent sample processing times

• Consider the polarization state of epithelial cells when interpreting results

How does Phospho-ERBB2 (Y1139) contribute to ERBB2-ERBB3 higher order complex signaling?

Recent research has revealed the importance of higher order ERBB receptor complexes beyond simple dimers:

Key Insights:

• ERBB2-ERBB3 heterodimers create potent mitogenic signals despite ERBB3's impaired kinase activity

• The phosphorylation mechanism of ERBB2 in these complexes challenges established models of phosphorylation in trans

• Higher order complexes facilitate "proxy phosphorylation" even at very low receptor levels (below 10,000 receptors per cell)

Experimental Evidence:

• An inhibitory RNA aptamer targeting ERBB3's extracellular domain disrupts higher order interactions without affecting heterodimerization

• This selective disruption inhibits ERBB2 phosphorylation and MAPK signaling while minimally affecting ERBB3 phosphorylation and AKT signaling

• This suggests a model of side-by-side oriented heterodimers that facilitate proxy phosphorylation

Research Applications:

• Use Phospho-ERBB2 (Y1139) antibody to:

  • Map the dynamics of higher order complex formation

  • Investigate how different therapeutic antibodies affect specific phosphorylation sites

  • Explore differences in signaling between canonical dimers and higher order complexes

What is the relationship between Phospho-ERBB2 (Y1139) and therapeutic resistance in HER2-positive cancers?

Understanding phosphorylation-specific resistance mechanisms is crucial for developing improved therapeutic strategies:

Clinical Correlations:

• Studies have found that phosphorylated HER2 status correlates with response to trastuzumab (Herceptin) therapy

• Acquisition of resistance to trastuzumab correlates with negativity for phosphorylated HER2 (p=0.028)

• Positive expression of phosphorylated HER2 may yield additional prognostic information in breast cancer

Molecular Mechanisms:

• Receptor Interactions:

  • Herceptin treatment prevents phosphorylation of tyrosine 1139 on apical ERBB2

  • Different phosphorylation sites may confer different sensitivities to therapeutic antibodies

  • Altered phosphorylation patterns may enable bypass signaling through alternative pathways

• Downstream Pathway Shifts:

  • Y1139 phosphorylation leads to Grb2 recruitment and can activate both:

    • Ras-dependent proliferation pathways

    • Ras-independent survival pathways

  • Shift to Ras-independent survival signaling may contribute to therapeutic resistance

Experimental Approach:
To investigate resistance mechanisms:

• Compare phosphorylation patterns between sensitive and resistant cells

• Assess changes in phosphorylation after acute vs. chronic treatment

• Determine whether resistance can be overcome by targeting specific phosphorylation-dependent pathways

How can Phospho-ERBB2 (Y1139) antibody be used to investigate spatial regulation of ERBB2 signaling?

Spatial organization of ERBB2 signaling is a critical dimension of its biological activity:

Spatial Regulation Evidence:

• Polarized epithelial cells show distinct phosphorylation patterns:

  • Apical ERBB2 is primarily phosphorylated at Y1139

  • Basolateral ERBB2 is predominantly phosphorylated at Y1248

• These different phosphorylation patterns activate distinct downstream signaling pathways

Methodological Approaches:

• Vectorial Biotinylation:

  • Selectively biotinylate either apical or basolateral membrane proteins

  • Pull down with streptavidin and immunoblot for Phospho-ERBB2 (Y1139)

  • Compare with other phosphorylation sites (e.g., Y1248)

  • Protocol details:

    1. Grow cells on permeable supports to establish polarity

    2. Apply biotin selectively to apical or basolateral compartments

    3. Lyse cells and capture biotinylated proteins

    4. Analyze by Western blot with phospho-specific antibodies

• Confocal Microscopy:

  • Perform immunofluorescence with Phospho-ERBB2 (Y1139) antibody

  • Counterstain with markers for different cellular compartments

  • Analyze in xy and xz planes to determine spatial distribution

  • Quantify colocalization with membrane domain markers

• Selective Inhibitor Application:

  • Apply Herceptin selectively to apical or basolateral domains

  • Monitor effects on different phosphorylation sites

  • Research shows apical Herceptin treatment inhibits Y1139 phosphorylation

Application Example:
Investigation of ERBB2 relocalization by mucin4 overexpression revealed that:

• ERBB2 relocalized by mucin4 overexpression is phosphorylated at both Y1248 and Y1139

• This dual phosphorylation may contribute to altered signaling profiles and therapeutic responses

How might single-cell analysis techniques enhance our understanding of Phospho-ERBB2 (Y1139) dynamics?

Traditional bulk analysis methods may mask important heterogeneity in ERBB2 phosphorylation:

Novel Methodological Approaches:

• Single-Cell Phospho-Proteomics:

  • Reveals cell-to-cell variability in phosphorylation patterns

  • May identify rare cell populations with unique phosphorylation signatures

  • Enables correlation of phosphorylation status with other cellular parameters

• Mass Cytometry (CyTOF):

  • Simultaneously measure multiple phosphorylation sites at single-cell resolution

  • Incorporate measurements of downstream signaling molecules

  • Create high-dimensional phosphorylation profiles of individual cells

• Live-Cell Phosphorylation Biosensors:

  • FRET-based sensors to monitor Y1139 phosphorylation in real-time

  • Track dynamics of phosphorylation in response to stimuli

  • Correlate with cellular behaviors (migration, division, death)

Research Applications:

• Investigate whether heterogeneous phosphorylation patterns predict therapeutic response

• Determine if rare cells with specific phosphorylation signatures drive disease progression

• Study temporal dynamics of phosphorylation/dephosphorylation cycles at single-cell level

What is the role of Phospho-ERBB2 (Y1139) in cancer stem cell maintenance and therapeutic resistance?

Emerging evidence suggests ERBB2 phosphorylation may play crucial roles in cancer stem cell (CSC) biology:

Research Questions:

• Do cancer stem cells display distinct ERBB2 phosphorylation patterns?

• Is Y1139 phosphorylation specifically involved in stemness maintenance?

• How do therapies targeting ERBB2 affect phosphorylation in CSC populations?

Experimental Approaches:

• Isolate CSCs using established markers (CD44+/CD24-, ALDH+)

• Compare phosphorylation patterns between CSCs and non-CSC populations

• Determine effects of manipulating Y1139 phosphorylation on:

  • Self-renewal capacity

  • Differentiation potential

  • Therapeutic resistance

Potential Mechanisms:

• Y1139 phosphorylation and Grb2 recruitment may activate specific survival pathways in CSCs

• Phosphorylation at Y1139 might regulate interactions with stemness-promoting factors

• CSCs may display altered spatial organization of ERBB2, affecting phosphorylation patterns

How can computational modeling incorporate Phospho-ERBB2 (Y1139) data to predict therapeutic responses?

Integrating phosphorylation data into predictive models represents a frontier in precision medicine:

Modeling Approaches:

• Dynamic Phosphorylation Networks:

  • Model the temporal sequence of phosphorylation events

  • Incorporate feedback and feedforward regulation

  • Simulate effects of targeted inhibitors on phosphorylation cascades

• Multi-scale Models:

  • Connect molecular phosphorylation events to cellular phenotypes

  • Integrate data across scales (molecular, cellular, tissue)

  • Predict emergent behaviors from phosphorylation patterns

• Machine Learning Applications:

  • Train algorithms on phosphorylation datasets to predict drug responses

  • Identify patterns not apparent through conventional analysis

  • Develop phosphorylation signatures that correlate with clinical outcomes

Data Integration Requirements:

• Quantitative phosphorylation measurements (absolute or relative)

• Temporal dynamics of multiple phosphorylation sites

• Spatial information about phosphorylation patterns

• Correlation with downstream pathway activation

• Clinical outcome data linked to phosphorylation profiles

Practical Applications:

• Predict which patients will benefit from anti-ERBB2 therapies

• Design optimal combination therapies based on phosphorylation profiles

• Develop treatment sequencing strategies to prevent resistance development