Phospho-ERBB3 (Y1222) Antibody

What is ERBB3/HER3 and why is phosphorylation at Y1222 significant?

ERBB3 (also known as HER3) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. This membrane-bound protein has a neuregulin binding domain but lacks an active kinase domain, meaning it can bind ligands but cannot convey signals through protein phosphorylation on its own . Instead, ERBB3 forms heterodimers with other EGF receptor family members that do possess kinase activity, leading to activation of pathways that promote cell proliferation or differentiation .

Phosphorylation at tyrosine 1222 (Y1222) is a critical event in cellular signaling pathways related to cell growth and survival. This specific phosphorylation site serves as a key point in the activation of downstream signaling cascades that contribute to cellular processes involved in both normal development and disease states, particularly cancer .

How specific is the Phospho-ERBB3 (Y1222) Antibody for this phosphorylation site?

The Phospho-ERBB3 (Y1222) rabbit monoclonal antibody exhibits high specificity for detecting phosphorylated ERBB3 at tyrosine 1222. This specificity is achieved through the use of a synthetic phosphorylated peptide around Y1222 of human ERBB3 (P21860) as the immunogen . The antibody has been developed using rabbit monoclonal technology, which typically provides higher specificity than polyclonal antibodies .

Validation experiments using phosphatase treatment demonstrate this specificity. When cell extracts are treated with calf intestinal phosphatase (CIP), the signal for phospho-ERBB3 Y1222 disappears in Western blot analysis, confirming the antibody is truly phospho-specific .

What applications can this antibody be used for?

The Phospho-ERBB3 (Y1222) antibody has been validated for the following applications:

While Western blot is the primary application for this antibody, allowing researchers to detect and quantify phosphorylated ERBB3 in cell and tissue lysates, ELISA applications may be suitable for specific experimental setups that require quantitative analysis of phospho-ERBB3 levels .

What species reactivity has been confirmed for this antibody?

The antibody has been primarily validated for human samples, with Cell Signaling's product also showing cross-reactivity with mouse samples . This makes it particularly valuable for cancer research using human cell lines and potentially for mouse models of cancer.

How does ERBB3 function in cancer signaling pathways?

ERBB3 is highly expressed in many cancer cells and activation of the ERBB3/PI3K pathway correlates with malignant phenotypes of adenocarcinomas . In tumor development, ERBB3 may function as an oncogenic unit together with other ErbB members. Research has demonstrated that ErbB2 requires ErbB3 to drive breast tumor cell proliferation, highlighting their cooperative role in oncogenesis .

The phosphorylation of ERBB3 at Y1222 is associated with activation of downstream signaling cascades that promote cell growth and survival, making it a focal point for investigating various cellular processes in cancer biology . By using the Phospho-ERBB3 (Y1222) antibody, researchers can explore:

• The molecular mechanisms of ERBB3 activation in different cancer types

• The relationship between ERBB3 phosphorylation and therapy resistance

• The interaction between ERBB3 and other members of the EGFR family in promoting tumor growth

• The potential of targeting ERBB3 phosphorylation for cancer treatment

Inhibiting interaction between ERBB3 and ErbB tyrosine kinases is viewed as a novel strategy for anti-tumor therapy .

How can I validate the specificity of Phospho-ERBB3 (Y1222) Antibody in my experimental system?

Validating antibody specificity is crucial for ensuring reliable results. For Phospho-ERBB3 (Y1222) antibody, consider the following approaches:

• Phosphatase treatment control: Treat cell lysates with calf intestinal phosphatase (CIP) as demonstrated in the ABClonal validation. This should eliminate the phospho-specific signal in Western blot .

• Stimulation experiments: Treat cells with neuregulin-1β (NRG1β/HRG1β), which is known to induce ERBB3 phosphorylation. The R&D Systems validation shows increased phosphorylation at Y1262 (another site) after NRG1β treatment, and similar approaches can be used for Y1222 .

• siRNA or CRISPR knockout: Depletion of ERBB3 protein should eliminate the signal detected by the phospho-specific antibody.

• Comparing total ERBB3 versus phospho-ERBB3: Run parallel Western blots with both total ERBB3 antibody and phospho-specific antibody to confirm that the detected band is indeed ERBB3.

• Peptide competition: Pre-incubation of the antibody with the phosphopeptide immunogen should block specific binding in your assay.

What are the technical considerations for preserving phosphorylation during sample preparation?

Preserving phosphorylation states during sample preparation is critical for accurate analysis with phospho-specific antibodies. Consider these methodological approaches:

• Rapid sample processing: Process samples quickly at cold temperatures (4°C) to minimize phosphatase activity.

• Phosphatase inhibitors: Include a robust cocktail of phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all lysis and processing buffers.

• Denaturation conditions: Use denaturing lysis buffers containing SDS to rapidly inactivate endogenous phosphatases.

• Avoid freeze-thaw cycles: Multiple freeze-thaw cycles can lead to protein degradation and loss of phosphorylation.

• Optimize lysis buffer: Different phosphorylation sites may be better preserved in specific buffer conditions. For ERBB3 Y1222, buffers containing 1% NP-40 or RIPA buffer with phosphatase inhibitors are commonly used.

• Consider direct lysis in SDS sample buffer: For particularly labile phosphorylation sites, direct lysis in hot SDS sample buffer can provide the best preservation of phosphorylation status.

How can I use this antibody to study ERBB3 signaling in response to different growth factors?

Growth factors like EGF and neuregulin can stimulate different phosphorylation states of HER receptors . To study these responses using the Phospho-ERBB3 (Y1222) antibody:

• Time-course experiments: Treat cells with appropriate growth factors (e.g., NRG1β, EGF) and harvest at different time points to analyze the kinetics of Y1222 phosphorylation.

• Dose-response studies: Vary the concentration of growth factors to determine threshold levels for ERBB3 phosphorylation.

• Co-treatment experiments: Combine growth factors with inhibitors of different EGFR family members to determine which receptors are responsible for transphosphorylation of ERBB3 at Y1222.

• Receptor heterodimerization analysis: Combine phospho-ERBB3 (Y1222) detection with immunoprecipitation of different EGFR family members to identify specific heterodimer pairs involved in signaling.

• Downstream signaling analysis: Correlate Y1222 phosphorylation with activation of downstream targets (e.g., AKT, MAPK) to understand signaling pathway integration.

What is the recommended Western blot protocol for Phospho-ERBB3 (Y1222) detection?

Based on manufacturer recommendations and validation data, the following Western blot protocol is suggested:

• Sample preparation:

  • Lyse cells in buffer containing phosphatase inhibitors

  • For 293T cells, use approximately 25μg protein per lane

• Gel electrophoresis and transfer:

  • Separate proteins on SDS-PAGE (note that ERBB3 runs at approximately 185 kDa)

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

• Blocking:

  • Block with 3-5% BSA in TBST (not milk, which contains phosphatases)

  • BSA at 3% concentration has been validated

• Primary antibody incubation:

  • Dilute Phospho-ERBB3 (Y1222) antibody to:

    • 1:500 - 1:2000 (ABClonal)

    • 1:1000 (Cell Signaling)

  • Incubate overnight at 4°C

• Secondary antibody:

  • HRP-conjugated anti-rabbit IgG secondary antibody

  • Typical dilution 1:10,000

• Detection:

  • ECL Enhanced Kit for visualization

  • Typical exposure time: 3 minutes, but adjust based on signal strength

What are common troubleshooting issues when using this antibody?

How can I quantify changes in ERBB3 Y1222 phosphorylation levels?

To quantitatively assess changes in ERBB3 Y1222 phosphorylation:

• Normalization approach:

  • Always run a total ERBB3 antibody on parallel samples or after stripping and reprobing

  • Calculate the ratio of phospho-ERBB3 to total ERBB3 to account for expression differences

• Loading control considerations:

  • Include housekeeping proteins (β-actin, GAPDH) as loading controls

  • For more precise quantification, consider normalizing to total protein using stain-free gels or Ponceau staining

• Densitometry recommendations:

  • Use image analysis software like ImageJ, Image Lab, or specialized quantification software

  • Ensure linear range of detection (avoid overexposed bands)

  • Perform at least three biological replicates for statistical analysis

• ELISA-based quantification:

  • For more precise quantification, consider sandwich ELISA approaches if higher throughput is needed

What positive controls are recommended for validating experimental results?

Based on the search results and literature, the following positive controls are recommended:

• Cell lines:

  • MDA-MB-453 human breast cancer cells treated with NRG1β (300 ng/mL for 5 minutes)

  • 293T cells expressing ERBB3

• Stimulation protocols:

  • Treatment with 300 ng/mL Recombinant Human NRG1-beta/HRG1-beta for 5 minutes

  • EGF stimulation in cells expressing both EGFR and ERBB3

• Negative controls:

  • Phosphatase treatment of lysates (CIP treatment, 20μL/400μL at 37°C for 1 hour)

  • Unstimulated cells that express ERBB3 but show minimal baseline phosphorylation

How can Phospho-ERBB3 (Y1222) Antibody be used in cancer research studies?

The Phospho-ERBB3 (Y1222) antibody enables multiple research approaches in cancer biology:

• Drug resistance mechanisms:

  • Monitor changes in ERBB3 Y1222 phosphorylation in response to EGFR or HER2-targeted therapies

  • Identify bypass signaling through ERBB3 as a mechanism of resistance to targeted therapies

• Biomarker development:

  • Correlate ERBB3 Y1222 phosphorylation levels with clinical outcomes in patient samples

  • Evaluate potential as a predictive biomarker for response to targeted therapies

• Combination therapy evaluation:

  • Assess inhibition of ERBB3 phosphorylation when combining different targeted agents

  • Test novel compounds targeting the ERBB3 signaling axis

• Heterodimer profiling:

  • Investigate which EGFR family members preferentially phosphorylate ERBB3 at Y1222 in different cancer types

  • Correlate heterodimer formation with downstream signaling activation

• Functional consequences:

  • Perform mutagenesis studies (Y1222F) to determine specific roles of this phosphorylation site

  • Connect Y1222 phosphorylation to specific downstream signaling events and phenotypic outcomes

What is the difference between Y1222 and other phosphorylation sites on ERBB3?

ERBB3 contains multiple tyrosine phosphorylation sites that serve different functions in signal transduction:

The Y1222 site is particularly important for oncogenic signaling, as activation of the ERBB3/PI3K pathway is correlated with malignant phenotypes of adenocarcinomas . The specific tyrosine residues that become phosphorylated determine which SH2-domain containing proteins can bind and which downstream pathways become activated.

How does heterodimer formation affect ERBB3 Y1222 phosphorylation?

Since ERBB3 lacks an active kinase domain, its phosphorylation at Y1222 depends on heterodimerization with other catalytically active EGFR family members:

• ERBB2-ERBB3 heterodimers:

  • ERBB2 (HER2) is the preferred dimerization partner for ERBB3

  • Research studies have demonstrated that in tumor development, ERBB2 requires ERBB3 to drive breast tumor cell proliferation

  • ERBB2-mediated phosphorylation of ERBB3 Y1222 is particularly important in HER2-positive breast cancers

• EGFR-ERBB3 heterodimers:

  • EGF can induce phosphorylation of ERBB3 through EGFR-ERBB3 heterodimers

  • The dynamics and extent of Y1222 phosphorylation may differ between EGFR-mediated and ERBB2-mediated phosphorylation

• Ligand-dependent dynamics:

  • NRG1β directly binds to ERBB3 and promotes its heterodimerization and phosphorylation

  • EGF binds to EGFR and can lead to transphosphorylation of ERBB3 through heterodimer formation

  • Different ligands may induce different patterns or kinetics of ERBB3 phosphorylation at Y1222

Understanding these heterodimerization patterns is crucial for developing therapeutic strategies targeting the ERBB network in cancer.

How can phosphatase treatment be optimized for antibody validation?

To properly validate the phospho-specificity of the Phospho-ERBB3 (Y1222) antibody using phosphatase treatment:

• Phosphatase selection:

  • Calf intestinal phosphatase (CIP) has been validated at 20μL/400μL of lysate, incubated at 37°C for 1 hour

  • Lambda phosphatase is an alternative with broad specificity for phospho-serine, phospho-threonine, and phospho-tyrosine

• Treatment protocol optimization:

  • Prepare parallel samples with and without phosphatase

  • Include phosphatase inhibitor controls to confirm specificity

  • Test different enzyme concentrations and incubation times for complete dephosphorylation

• Buffer considerations:

  • Ensure buffer compatibility with phosphatase activity

  • Remove detergents that might inhibit phosphatase activity through dialysis or dilution if necessary

• Validation by immunoblotting:

  • Run treated and untreated samples side by side

  • Probe with both phospho-specific and total ERBB3 antibodies to confirm that only the phospho-signal is eliminated

This methodological approach provides strong evidence for the phospho-specificity of the antibody and should be included as a control in publications using this antibody for the first time in a new experimental system.

What are emerging applications for Phospho-ERBB3 (Y1222) Antibody in precision medicine?

The Phospho-ERBB3 (Y1222) antibody has potential applications in precision medicine research:

• Patient stratification:

  • Identifying patients whose tumors show high levels of ERBB3 Y1222 phosphorylation

  • Correlating phosphorylation levels with response to targeted therapies

• Resistance monitoring:

  • Tracking changes in ERBB3 phosphorylation during treatment

  • Detecting emergence of ERBB3-dependent bypass signaling as a resistance mechanism

• Combination therapy design:

  • Rational design of combination therapies targeting multiple EGFR family members

  • Monitoring on-target efficacy of novel ERBB3-directed therapeutics

What technical advances might improve detection of phosphorylated ERBB3?

Future technical developments that could enhance research using phospho-ERBB3 antibodies include:

• Single-cell phospho-protein analysis:

  • Adapting phospho-ERBB3 detection for mass cytometry or imaging mass cytometry

  • Developing protocols for single-cell Western blotting with phospho-specificity

• Multiplexed detection systems:

  • Simultaneous detection of multiple phosphorylation sites on ERBB3 and other EGFR family members

  • Integration with other signaling pathway components for systems-level analysis

• Live-cell imaging:

  • Development of phospho-specific intrabodies or biosensors for real-time monitoring of ERBB3 phosphorylation

• Improved tissue analysis:

  • Optimization of immunohistochemistry protocols for phospho-ERBB3 detection in FFPE tissues

  • Quantitative imaging approaches for phospho-protein analysis in tumor samples