Phospho-ERBB3 (Y1328) Antibody

What is the ERBB3 receptor and what cellular processes does phosphorylation at Y1328 regulate?

ERBB3 (also known as HER3) functions as a tyrosine-protein kinase that serves as a cell surface receptor for neuregulins. The receptor plays an essential role in signal transduction pathways, particularly when phosphorylated. Specifically, Y1328 phosphorylation represents one of several key tyrosine phosphorylation sites that may contribute to downstream pathway activation. ERBB3 binds to neuregulin-1 (NRG1), which increases phosphorylation on tyrosine residues and promotes its association with the p85 subunit of phosphatidylinositol 3-kinase. Additionally, ERBB3 may be activated by CSPG5 and is involved in the regulation of myeloid cell differentiation .

What detection methods are compatible with Phospho-ERBB3 (Y1328) antibodies?

Phospho-ERBB3 (Y1328) antibodies are compatible with multiple detection methodologies depending on the specific research question. Validated applications include:

• Western Blotting (WB) for protein expression analysis

• Immunohistochemistry on paraffin-embedded sections (IHC-P)

• Immunocytochemistry/Immunofluorescence (ICC/IF) for cellular localization

• ELISA at recommended dilutions of 1:5000

• In situ Proximity Ligation Assay (PLA) when used as part of an antibody pair

For IHC applications, a dilution range of 1:100-1:300 is typically recommended to achieve optimal signal-to-noise ratio .

What are the optimal storage and handling conditions for Phospho-ERBB3 (Y1328) antibodies?

To maintain antibody functionality, Phospho-ERBB3 (Y1328) antibodies should be stored at -20°C or lower (-80°C for long-term storage). The antibody should be aliquoted upon receipt to avoid repeated freeze-thaw cycles that can significantly compromise antibody activity and specificity. Most commercial preparations are supplied in PBS buffer containing preservatives such as 50% glycerol, 0.5% BSA, and 0.02% sodium azide to maintain stability . When working with the antibody, it's advisable to thaw aliquots completely before use and return them to cold storage immediately after application to preserve antibody integrity.

How should positive and negative controls be designed for Phospho-ERBB3 (Y1328) antibody experiments?

Robust experimental design requires appropriate controls. For Phospho-ERBB3 (Y1328) antibody experiments:

Positive controls:

• Human breast cancer cell lines known to express high levels of phosphorylated ERBB3, particularly those with PIK3CA mutations (e.g., T47D cells with H1047R mutation)

• Cells stimulated with neuregulin-1 (NRG1) to induce ERBB3 phosphorylation

• Paired samples using both phospho-specific and total ERBB3 antibodies

Negative controls:

• Cell lines with HER3 knockdown via shRNA or CRISPR

• Phosphatase treatment of samples to remove phosphorylation

• Competing peptide blocking experiments using synthetic phosphopeptides matching the Y1328 region

• Isotype control antibodies to assess non-specific binding

Importantly, research has shown limitations in commercially available phospho-Y1328 antibodies, with some lacking sensitivity and specificity, underscoring the need for thorough validation in your experimental system .

What are the recommended sample preparation protocols to preserve phosphorylation status at Y1328?

Preserving phosphorylation status requires careful sample handling:

• For cell lysates:

  • Harvest cells rapidly on ice

  • Use lysis buffers containing phosphatase inhibitors (sodium orthovanadate, sodium fluoride, β-glycerophosphate)

  • Process samples at 4°C to minimize enzymatic activity

  • Include protease inhibitors to prevent protein degradation

• For tissue samples:

  • Flash-freeze tissue immediately after collection

  • Use phosphatase inhibitor-containing fixatives

  • Process and embed tissues rapidly to minimize dephosphorylation

  • Consider preparation methods that allow dual recognition techniques such as PLA

• For both:

  • Avoid multiple freeze-thaw cycles

  • Standardize time between collection and fixation/lysis

How can Proximity Ligation Assay (PLA) be optimized for detecting ERBB3 Y1328 phosphorylation in situ?

The Proximity Ligation Assay offers superior sensitivity for detecting phosphorylated ERBB3 at Y1328 by using antibody pairs:

• Antibody selection and optimization:

  • Use a rabbit polyclonal antibody against phospho-ERBB3 Y1328 (recommended dilution 1:1200)

  • Pair with a mouse monoclonal antibody against total ERBB3 (recommended dilution 1:50)

  • Validate antibody specificity individually before performing PLA

• Protocol considerations:

  • Perform stringent blocking to minimize background

  • Optimize incubation times for primary antibodies

  • Ensure appropriate fixation (typically 4% paraformaldehyde followed by permeabilization)

  • Use BlobFinder software (available from The Centre for Image Analysis at Uppsala University) for quantitative analysis

• Data interpretation:

  • Each red dot in PLA represents a single phosphorylated protein instance

  • Compare signal intensity across multiple fields and biological replicates

  • Include appropriate negative controls to determine background signal levels

What factors contribute to variability in Phospho-ERBB3 (Y1328) antibody performance across experimental systems?

Several factors can affect antibody performance:

• Antibody characteristics:

  • Production method (monoclonal vs. polyclonal)

  • Host species and purification methods

  • Phospho-epitope recognition specificity

  • Lot-to-lot variability in commercial antibodies

• Experimental variables:

  • Fixation methods and duration

  • Antigen retrieval protocols (particularly critical for IHC)

  • Buffer composition and pH

  • Protein conformation affecting epitope accessibility

• Biological factors:

  • Expression levels of total ERBB3 protein

  • Dynamic nature of phosphorylation (transient vs. stable)

  • Potential cross-reactivity with other ERBB family members

  • Cell/tissue-specific post-translational modifications

Published research indicates variability in commercial antibody performance, with some lacking sufficient sensitivity and specificity for certain applications, necessitating thorough validation before use in critical experiments .

How does ERBB3 Y1328 phosphorylation influence PI3K/AKT versus MAPK pathway activation?

Research using isogenic cell lines has revealed differential roles for ERBB3 Y1328 phosphorylation in signaling pathway activation:

• PI3K/AKT pathway:

  • HER3 knockdown consistently reduces AKT phosphorylation across both E545K and H1047R PIK3CA mutant cells

  • This suggests that ERBB3 signaling is crucial for PI3K pathway activation regardless of the specific PIK3CA mutation

• MAPK pathway:

  • HER3 knockdown reduces ERK phosphorylation in H1047R cells but not in E545K cells

  • This indicates a differential dependency on ERBB3 for MAPK pathway activation based on the specific oncogenic PIK3CA mutation

  • H1047R mutations appear to rely more heavily on HER3 signaling for both PI3K and MAPK pathway activation

• Phosphorylation patterns:

  • Phosphoproteomic analysis demonstrates different patterns of Y1328 phosphorylation between E545K and H1047R isogenic cell lines

  • H1047R cells show increased phosphorylation at Y1328 compared to E545K cells

  • These differences may explain the differential dependency on ERBB3 signaling

What methodological approaches can distinguish Y1328 phosphorylation from other ERBB3 phosphorylation sites?

Differentiating between ERBB3 phosphorylation sites requires specialized techniques:

• Mass spectrometry-based approaches:

  • Phosphoproteomics using SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture)

  • Multiple Reaction Monitoring (MRM) for targeted phosphopeptide quantification

  • Phospho-enrichment strategies using titanium dioxide or IMAC

• Antibody-based methods:

  • Site-specific phospho-antibodies with validated specificity

  • In situ proximity ligation assay to detect specific phosphorylation events

  • Phospho-flow cytometry for single-cell analysis

• Genetic approaches:

  • Site-directed mutagenesis (Y1328F) to abolish phosphorylation at this specific site

  • Correlation with functional outcomes to determine site-specific effects

• Computational analysis:

  • Phosphosite prediction algorithms to identify potential kinases

  • Structural modeling to understand accessibility of specific sites

Research has demonstrated that different phosphorylation sites on the same protein can be differentially regulated, as evidenced by contrasting patterns between Y1289 and Y1328 phosphorylation in response to PIK3CA mutations .

How does ERBB3 Y1328 phosphorylation status differ across cancer types and genetic backgrounds?

Phosphorylation of ERBB3 at Y1328 shows notable variation across cancer contexts:

• PIK3CA mutational status influence:

  • H1047R (kinase domain) mutations show increased Y1328 phosphorylation compared to E545K (helical domain) mutations

  • This differential phosphorylation pattern has been observed in both engineered isogenic cell lines and actual breast cancer cell lines (T47D vs. MCF-7)

  • The mechanism behind this differential phosphorylation remains under investigation

• Breast cancer subtypes:

  • Different ERBB3 phosphorylation patterns may contribute to the unique biological behaviors observed across breast cancer subtypes

  • Phosphorylation status correlates with dependency on specific signaling pathways

• Growth factor independence:

  • HER3 knockdown has a profound effect on growth factor-independent proliferation in H1047R cells

  • This suggests that Y1328 phosphorylation may contribute to oncogenic potential through altered cellular dependency relationships

What methodological considerations are important when using Phospho-ERBB3 (Y1328) antibodies to evaluate therapeutic responses?

When assessing therapeutic responses using Phospho-ERBB3 (Y1328) antibodies:

• Timing of sample collection:

  • Phosphorylation events are dynamic and can change rapidly after drug treatment

  • Establish time-course experiments to capture both immediate and delayed effects

  • Consider the pharmacokinetics of the therapeutic agent being evaluated

• Quantification methods:

  • Normalize phospho-signals to total ERBB3 protein levels

  • Use digital image analysis for IHC or IF to obtain quantitative data

  • Consider multiple phosphorylation sites to build a more complete picture of ERBB3 activation status

• Integration with functional assays:

  • Correlate changes in Y1328 phosphorylation with downstream pathway activity (AKT, ERK)

  • Connect phosphorylation changes to functional outcomes (proliferation, survival, migration)

  • Use genetic manipulation (knockdown/reexpression) to validate antibody findings

• Technical validation:

  • Include appropriate positive controls (e.g., neuregulin-stimulated cells)

  • Use multiple detection methods when possible (Western blot, IHC, PLA)

  • Be aware of potential limitations in antibody specificity across different applications

What are the most common technical challenges when working with Phospho-ERBB3 (Y1328) antibodies and how can they be addressed?

Researchers commonly encounter these challenges:

• Low signal-to-noise ratio:

  • Optimize antibody concentration through titration experiments

  • Increase blocking stringency (5% BSA or 5% milk in TBS-T)

  • For Western blots, consider membrane transfer conditions and blocking time

  • For IHC/IF, optimize antigen retrieval methods and detection systems

• Non-specific binding:

  • Include appropriate negative controls (isotype antibodies, competing peptides)

  • Pre-absorb antibodies with non-specific proteins

  • Increase washing steps in duration and number

  • Use more selective secondary antibodies

• Phosphorylation instability:

  • Ensure phosphatase inhibitors are fresh and at appropriate concentrations

  • Standardize time between sample collection and processing

  • Consider using phosphatase treatment as a negative control

  • Process samples at 4°C whenever possible

• Antibody specificity concerns:

  • Validate using knockout or knockdown samples

  • Confirm specificity using phosphopeptide competition

  • Validate results using orthogonal methods (mass spectrometry)

  • Be aware that commercial antibodies may lack sufficient sensitivity for certain applications

How can researchers validate the specificity of Phospho-ERBB3 (Y1328) antibodies in their experimental systems?

Rigorous validation approaches include:

• Genetic validation:

  • Use ERBB3 knockdown or knockout cells as negative controls

  • Create Y1328F point mutation to specifically eliminate this phosphorylation site

  • Compare results across multiple cell lines with known ERBB3 expression profiles

• Biochemical validation:

  • Perform peptide competition assays using phosphorylated and non-phosphorylated Y1328 peptides

  • Conduct lambda phosphatase treatment of samples to remove phosphorylation

  • Compare results using multiple antibodies targeting the same phosphosite from different vendors

• Correlation with biological stimuli:

  • Assess phosphorylation changes following neuregulin-1 stimulation

  • Evaluate phosphorylation changes after treatment with kinase inhibitors

  • Compare to other ERBB3 phosphorylation sites (e.g., Y1289) using site-specific antibodies

• Cross-platform validation:

  • Confirm findings using complementary techniques (Western blot, IHC, PLA, mass spectrometry)

  • Compare with published phosphoproteomic datasets

  • Consider dual-recognition strategies such as proximity ligation assays to improve specificity