Phospho-EGFR (Ser1071) Antibody

What is the biological significance of EGFR Ser1071 phosphorylation?

EGFR Ser1071 phosphorylation represents a key post-translational modification in the epidermal growth factor receptor signaling pathway. This specific phosphorylation occurs within a serine/threonine-rich region of the receptor that has been implicated in receptor trafficking and internalization. Research indicates that phosphorylation at Ser1071 accumulates more slowly after EGF stimulation compared to other phosphorylation sites involved in immediate signal transduction, suggesting its role in receptor regulation rather than initial signal propagation . The temporal dynamics of Ser1071 phosphorylation correlate with receptor internalization processes, indicating its potential involvement in the spatial-temporal control of EGFR trafficking and downstream signaling regulation.

How do I select the appropriate application when using Phospho-EGFR (Ser1071) antibodies?

Selection should be based on your specific research question and experimental design:

For detecting physiological changes in Ser1071 phosphorylation, Western blotting is recommended due to its ability to distinguish specific bands at the expected molecular weight (150-160 kDa) . For quantitative high-throughput analysis, particularly when evaluating inhibitor effects or stimulation conditions across multiple samples, ELISA-based methods provide better standardization .

What are the recommended storage conditions for Phospho-EGFR (Ser1071) antibodies?

Most Phospho-EGFR (Ser1071) antibodies require careful storage to maintain specificity and activity:

• Store at -20°C for long-term storage (up to one year)

• For frequent use, store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can degrade antibody performance

• Most commercial antibodies are supplied in buffers containing glycerol (typically 50%), which prevents freezing at -20°C

• Standard buffer composition includes phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide and 50% glycerol

Upon receipt of a new antibody, it is advisable to aliquot the stock solution to minimize freeze-thaw cycles if the antibody will be used for multiple experiments over time.

How can I validate the specificity of a Phospho-EGFR (Ser1071) antibody for my experimental system?

Validation should include multiple complementary approaches:

• Positive and negative controls:

  • Positive control: Treat cells with EGF (25 ng/mL for 5-15 minutes) to induce EGFR phosphorylation

  • Negative control: Use EGFR inhibitors (e.g., PD168393) to block phosphorylation

• Phosphatase treatment control:

  • Treat one sample with lambda phosphatase to remove phosphorylation

  • Compare with untreated sample - signal should disappear in phosphatase-treated sample

• Peptide competition assay:

  • Pre-incubate antibody with phospho-peptide containing the Ser1071 site (Y-S-S(p)-D-P)

  • Signal should be blocked by phospho-peptide but not by non-phosphorylated peptide

• Cross-reactivity testing:

  • Test against recombinant proteins of related receptor family members (e.g., ErbB2/HER2, ErbB3, ErbB4)

  • A specific antibody should recognize only EGFR and not other family members

ELISA competition assays have demonstrated that recombinant extracellular domains of EGFR, but not related receptors (ErbB2, ErbB3, or ErbB4), compete for binding with Phospho-EGFR antibodies, confirming specificity .

What normalization methods should be used when analyzing Phospho-EGFR (Ser1071) data?

Multiple normalization approaches are recommended for robust quantitative analysis:

When analyzing temporal dynamics of phosphorylation, normalization to total EGFR is particularly important as receptor levels may change due to degradation following EGF stimulation .

How does p38 MAPK signaling regulate EGFR Ser1071 phosphorylation and what methods can detect this relationship?

The p38 MAPK pathway plays a critical role in stress-induced EGFR internalization and recycling, with evidence suggesting direct involvement in Ser1071 phosphorylation:

• Research has identified that EGF-induced phosphorylation at sites near Ser1071 (specifically Ser1039 and Thr1041) is blocked by SB-202190, a selective p38 inhibitor

• This suggests coordinated phosphorylation involving Ser1071 and nearby residues in controlling EGFR trafficking

Experimental approach to investigate this relationship:

• Pharmacological inhibition:

  • Pretreat cells with SB-202190 (p38 inhibitor) at 1-10 μM for 30-60 minutes

  • Stimulate with EGF (25 ng/mL)

  • Analyze Ser1071 phosphorylation by Western blot or ELISA

• Genetic manipulation:

  • Use siRNA knockdown of p38α/β MAPK

  • Alternatively, express dominant-negative p38 constructs

  • Compare Ser1071 phosphorylation in control vs. p38-depleted cells

• Quantitative analysis:

  • Apply Selected Reaction Monitoring (SRM) mass spectrometry to precisely quantify phosphorylation at multiple sites simultaneously

  • This technique provides higher specificity than antibody-based methods

What are the temporal dynamics of EGFR Ser1071 phosphorylation and how does it compare with other phosphorylation sites?

EGFR phosphorylation occurs with distinct temporal patterns at different sites:

Research using temporal profiling revealed that Ser1071 phosphorylation accumulates more slowly than tyrosine sites involved in immediate signaling events, suggesting its role in receptor downregulation rather than initial signal propagation .

To investigate these dynamics:

• Stimulate cells with EGF (25 ng/mL)

• Collect samples at multiple timepoints (0, 1, 5, 10, 15, 30, 60 minutes)

• Analyze using either:

  • Western blotting with phospho-specific antibodies

  • Selected Reaction Monitoring mass spectrometry

  • Phospho-EGFR ELISAs with temporal sampling

How can Cell-Based ELISA systems be optimized for studying Phospho-EGFR (Ser1071) in various experimental conditions?

Cell-Based ELISA systems offer advantages for studying EGFR phosphorylation in intact cells. Optimization strategies include:

• Cell density optimization:

  • Determine optimal seeding density (typically >5000 cells per well)

  • Ensure even distribution across the plate

  • Allow sufficient time for adherence before treatments

• Treatment conditions:

  • Serum-starve cells (0.1-0.5% serum) for 12-24 hours before stimulation

  • For EGF stimulation, use 10-50 ng/mL for 5-15 minutes

  • For inhibitor studies, pretreat cells before EGF addition

• Multiple normalization methods implementation:

  • Include wells for total EGFR detection

  • Include wells for housekeeping protein (GAPDH) detection

  • Use Crystal Violet staining to normalize for cell number

• Validation across multiple cell lines:

  • Compare responses in cells with different EGFR expression levels

  • Include positive control cell lines (e.g., A431 with high EGFR expression)

The qualitative nature of Cell-Based ELISAs makes multiple normalization approaches particularly important for reliable data interpretation .

How can Phospho-EGFR (Ser1071) antibodies be used in cancer research studies?

Phospho-EGFR (Ser1071) antibodies provide valuable tools for investigating aberrant EGFR signaling in cancer:

• Mechanisms of EGFR-targeted therapy resistance:

  • Compare Ser1071 phosphorylation patterns in sensitive vs. resistant cell lines

  • Analyze changes after treatment with EGFR tyrosine kinase inhibitors (TKIs)

  • Investigate whether Ser1071 phosphorylation persists when tyrosine phosphorylation is inhibited

• Receptor trafficking alterations in cancer:

  • Assess whether altered Ser1071 phosphorylation correlates with abnormal receptor recycling

  • Compare primary vs. metastatic tumor samples for differences in phosphorylation patterns

• Combination with other biomarkers:

  • Multiplex analysis with other EGFR phosphorylation sites

  • Correlate with downstream signaling markers (ERK, AKT, STAT phosphorylation)

  • Investigate relationship with p38 MAPK activity in tumor samples

Over-expression of EGFR has been reported in tumors of breast, lung, colon, cervix, ovary, esophagus and endometrium, making phosphorylation analysis relevant across multiple cancer types .

What role does Ser1071 phosphorylation play in EGFR internalization and trafficking, and how can this be experimentally demonstrated?

The serine/threonine-rich region of EGFR containing Ser1071 is implicated in receptor internalization and trafficking:

• Evidence for involvement in endocytosis:

  • Phosphorylation at Ser1071 occurs with similar kinetics as Tyr998, which is known to be critical for endocytosis

  • Both sites are phosphorylated more slowly than sites involved in immediate signal transduction

• Experimental approaches to study Ser1071 in trafficking:

  a) Site-directed mutagenesis:

  • Generate S1071A (phospho-deficient) EGFR mutants

  • Express in EGFR-null cells

  • Compare internalization rates with wild-type EGFR

  b) Internalization assays:

  • Surface biotinylation of EGFR followed by internalization tracking

  • Flow cytometry-based endocytosis assays using fluorescently-labeled EGF

  • Confocal microscopy to visualize receptor trafficking

  c) Co-immunoprecipitation studies:

  • Investigate whether Ser1071 phosphorylation affects binding to endocytic adaptor proteins

  • Compare wild-type and S1071A mutant EGFR

• Cross-talk with other phosphorylation sites:

  • Investigate whether mutation at Ser1071 affects phosphorylation at nearby sites (Ser1039, Thr1041)

  • Assess whether this creates compensatory changes in receptor trafficking pathways

How do different stimuli affect EGFR Ser1071 phosphorylation compared to classic EGF stimulation?

EGFR phosphorylation can be induced by diverse stimuli beyond EGF, with potentially distinct patterns:

• Growth factor comparisons:

  • Compare EGF with other EGFR ligands (TGF-α, amphiregulin, betacellulin, epiregulin, HB-EGF)

  • Analyze whether different ligands induce distinct patterns of Ser1071 phosphorylation

• Stress and cytokine stimulation:

  • TNF-α, UV irradiation, and chemotherapeutic agents (e.g., cisplatin) activate p38 MAPK pathways

  • These stimuli induce EGFR internalization through mechanisms potentially involving serine/threonine phosphorylation

  • Compare Ser1071 phosphorylation induced by these stressors vs. EGF

• Transactivation pathways:

  • G-protein coupled receptor agonists can transactivate EGFR

  • Compare Ser1071 phosphorylation following GPCR activation vs. direct EGF stimulation

Experimental protocol for comparative analysis:

• Serum-starve cells for 16-24 hours

• Treat with various stimuli:

  • EGF (25 ng/mL)

  • Other EGFR ligands (25-50 ng/mL)

  • TNF-α (10-50 ng/mL)

  • UV irradiation (10-50 J/m²)

  • GPCR agonists (e.g., angiotensin II, 100 nM)

• Collect samples at multiple timepoints (5-60 minutes)

• Analyze Ser1071 phosphorylation by Western blot or ELISA

• Compare temporal dynamics and magnitude of phosphorylation

What are emerging technologies for studying dynamic phosphorylation events at EGFR Ser1071?

Several cutting-edge technologies are advancing our ability to study phosphorylation dynamics:

• Proximity ligation assays (PLA):

  • Allows visualization of specific phosphorylation events in situ

  • Provides spatial information about where in the cell phosphorylation occurs

  • Can detect interactions between phosphorylated EGFR and binding partners

• FRET-based biosensors:

  • Genetically encoded sensors can monitor EGFR phosphorylation in real-time

  • Enables visualization of phosphorylation/dephosphorylation dynamics in living cells

  • Could be developed for Ser1071-specific detection

• Mass spectrometry advances:

  • Selected/Multiple Reaction Monitoring (SRM/MRM) for precise quantification

  • Parallel Reaction Monitoring (PRM) for improved specificity

  • These techniques allow multiplexed analysis of multiple phosphorylation sites simultaneously

• Single-cell phosphoproteomics:

  • Emerging technology to analyze phosphorylation events at single-cell resolution

  • Could reveal heterogeneity in EGFR phosphorylation responses within populations

How does Ser1071 phosphorylation integrate with the broader network of EGFR post-translational modifications?

EGFR function is regulated by complex, interconnected post-translational modifications:

• Cross-talk with tyrosine phosphorylation:

  • Investigate whether Ser1071 phosphorylation affects nearby tyrosine phosphorylation sites

  • Determine if tyrosine kinase inhibitors (TKIs) indirectly affect Ser1071 phosphorylation

• Integration with ubiquitination:

  • Examine how Ser1071 phosphorylation relates to receptor ubiquitination

  • Determine whether it affects binding of E3 ubiquitin ligases (e.g., Cbl)

• Interplay with glycosylation:

  • Study how receptor glycosylation affects accessibility of Ser1071 for phosphorylation

  • Determine if glycosylation patterns affect kinase recognition of this site

• Multi-site phosphorylation patterns:

  • Apply phosphoproteomics to map the temporal sequence of phosphorylation events

  • Identify potential "phosphorylation barcodes" that direct specific receptor fates