Phospho-PEA15 (S104) Antibody

What is the functional significance of PEA15 phosphorylation at S104?

Phosphorylation of PEA15 at serine 104 plays a crucial role in regulating its interactions with other proteins, particularly ERK1/2. When PEA15 is phosphorylated at S104, it loses its ability to bind ERK1/2, thereby allowing ERK1/2 to translocate to the nucleus and activate transcription factors . This phosphorylation is one of two critical modifications (the other being at S116) that together act as a molecular switch, potentially converting PEA15 from a tumor suppressor to a tumor promoter . Importantly, S104 phosphorylation is mediated by protein kinase C (PKC), while S116 can be phosphorylated by calcium-calmodulin-dependent protein kinase II or AKT/PKB .

What are the recommended applications for Phospho-PEA15 (S104) antibodies?

Phospho-PEA15 (S104) antibodies have been validated for multiple applications:

Researchers should optimize the antibody concentration for their specific experimental conditions and sample types .

How should Phospho-PEA15 (S104) antibodies be stored to maintain reactivity?

For optimal antibody performance, Phospho-PEA15 (S104) antibodies should be stored at -20°C for up to one year from the date of receipt . Many commercial preparations come in a stabilizing buffer containing 50% glycerol to prevent freeze-thaw damage . It's advisable to prepare small aliquots to minimize freeze-thaw cycles, which can degrade antibody quality. Some suppliers recommend 4°C for short-term storage after initial use .

What species reactivity can be expected with Phospho-PEA15 (S104) antibodies?

Most commercially available Phospho-PEA15 (S104) antibodies demonstrate cross-reactivity with:

The high sequence conservation of the phosphorylation site across species enables this broad reactivity .

What are the optimal sample preparation techniques for detecting phospho-PEA15 (S104) in Western blotting?

For optimal detection of phosphorylated PEA15 at S104 in Western blotting:

• Immediate sample processing: Process tissue or cell samples immediately after collection to preserve phosphorylation status.

• Phosphatase inhibitors: Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate, and phosphatase inhibitor cocktails) in your lysis buffer to prevent dephosphorylation during sample preparation .

• Protein extraction: Use a lysis buffer containing 1% Triton X-100, 150mM NaCl, 10mM sodium phosphate buffer (pH 7.0), 1mM DTT, and protease/phosphatase inhibitors .

• Sample handling: Keep samples on ice throughout processing and avoid repeated freeze-thaw cycles.

• Loading controls: Use both total PEA15 antibody and a housekeeping protein control in parallel samples to normalize phosphorylation levels .

• Expected molecular weight: Look for a band at approximately 15 kDa, which corresponds to the molecular weight of PEA15 .

How can I validate the specificity of a Phospho-PEA15 (S104) antibody?

To validate the specificity of a Phospho-PEA15 (S104) antibody:

• Phosphatase treatment: Treat half of your sample with lambda phosphatase to remove phosphate groups. A specific phospho-antibody should show decreased or absent signal in the phosphatase-treated sample.

• Peptide competition: Pre-incubate the antibody with the phosphorylated peptide used as the immunogen (often available from the antibody manufacturer). This should abolish specific binding.

• Positive controls: Include samples known to have high levels of PEA15 phosphorylation, such as C6 cells treated with PMA, which has been documented as a positive control for S104 phosphorylation .

• Phosphomimetic mutants: If feasible, use cell lysates expressing PEA15 with S104D mutation (phosphomimetic) and S104A mutation (non-phosphorylatable) as positive and negative controls, respectively .

• Knockout/knockdown validation: Use samples from PEA15 knockout or knockdown models to confirm antibody specificity.

What is the best approach for quantifying phospho-PEA15 (S104) levels in cell-based assays?

For quantitative analysis of phospho-PEA15 (S104) levels:

• Cell-Based ELISA: Use commercially available colorimetric ELISA kits specifically designed for phospho-PEA15 (S104) detection. These assays provide high sensitivity and allow for normalization to total PEA15 levels .

• Western blot densitometry: For semi-quantitative analysis, perform Western blotting with both phospho-specific and total PEA15 antibodies on parallel samples, then calculate the ratio of phosphorylated to total protein using densitometry software.

• Multiplexed assays: Consider phospho-protein array technologies that allow simultaneous detection of multiple phosphorylation sites if examining broader signaling networks.

• Sample normalization: Always normalize phospho-PEA15 (S104) levels to total PEA15 expression to account for variations in total protein levels between samples.

• Time-course experiments: PEA15 phosphorylation is dynamic; consider examining multiple time points after stimulus application to capture the full phosphorylation profile.

How does phosphorylation at S104 interact with S116 phosphorylation to regulate PEA15 function?

The interplay between S104 and S116 phosphorylation creates a complex regulatory system:

• Hierarchical phosphorylation: Research suggests that phosphorylation at S104 (by PKC) may facilitate subsequent phosphorylation at S116 (by CaMKII or AKT) .

• Functional switch: Double phosphorylation at both S104 and S116 appears to switch PEA15 from a tumor suppressor to a tumor promoter role . In ovarian cancer studies, tissues from patients were significantly more likely to express PEA15 phosphorylated at both sites compared to adjacent normal tissues .

• Structural changes: NMR spectroscopy studies indicate that phosphorylation of PEA15 induces conformational changes that affect its death effector domain (DED), which is critical for protein-protein interactions .

• Differential binding partners: While unphosphorylated PEA15 preferentially binds ERK1/2, double-phosphorylated PEA15 (at both S104 and S116) shows enhanced binding to FADD and caspase-8, thereby inhibiting apoptosis .

• Mutant studies: Phosphomimetic (S104D/S116D, PEA15-DD) and non-phosphorylatable (S104A/S116A, PEA15-AA) mutants have demonstrated that the non-phosphorylatable form has more potent antitumorigenic effects in ovarian cancer, partially through inhibition of β-catenin expression and nuclear translocation .

What are the most effective experimental designs for studying PEA15 phosphorylation dynamics in response to stimuli?

For investigating dynamic phosphorylation of PEA15:

• Time-course experiments: Collect samples at multiple time points (e.g., 0, 5, 15, 30, 60 minutes, 2, 6, 24 hours) after stimulus application to capture the full phosphorylation kinetics.

• Kinase inhibitor studies: Use specific inhibitors of PKC, CaMKII, or AKT/PKB to determine their relative contributions to S104 and S116 phosphorylation under different conditions .

• Phosphatase inhibitor studies: Employ specific phosphatase inhibitors to understand the rate of dephosphorylation and identify the phosphatases involved.

• Cellular fractionation: Combine with subcellular fractionation to track how phosphorylation status affects PEA15 localization and its interaction partners in different cellular compartments.

• Proximity ligation assays: Use this technique to visualize and quantify interactions between phospho-PEA15 and its binding partners like ERK1/2 or FADD in situ.

• Phosphoproteomic analysis: Apply mass spectrometry-based phosphoproteomics to identify novel phosphorylation sites or interaction partners of PEA15 under different experimental conditions.

• Live-cell imaging: Consider using phospho-specific biosensors if available to monitor PEA15 phosphorylation in real-time in living cells.

What are the critical considerations when interpreting discrepancies between different phospho-PEA15 (S104) detection methods?

When facing contradictory results across different detection methods:

• Antibody epitope differences: Different antibodies may recognize slightly different epitopes surrounding the phosphorylated S104 site, affecting their sensitivity and specificity. Review the immunogen sequence information provided by manufacturers .

• Sample preparation variations: Phosphorylation can be lost during sample preparation if phosphatase inhibitors are insufficient or if samples are processed slowly. Different methods have different sample preparation requirements that might affect phosphorylation preservation.

• Detection sensitivity thresholds: Western blotting, IHC, and ELISA have different detection limits. ELISA is generally more sensitive for quantification, while Western blotting provides information about protein size and potential cross-reactivity .

• Spatial resolution considerations: IHC provides information about cellular and subcellular localization of phosphorylated proteins that may be lost in lysate-based methods like Western blotting.

• Normalization strategy: Different normalization approaches (to total protein, housekeeping proteins, or cell number) can yield apparently contradictory results. Always report how normalization was performed.

• Context-dependent phosphorylation: PEA15 phosphorylation is highly context-dependent and can vary with cell type, culture conditions, and stimulation parameters .

How can Phospho-PEA15 (S104) antibodies be utilized to investigate disease-specific alterations in PEA15 function?

For disease-specific applications:

• Cancer research: Use phospho-PEA15 (S104) antibodies in tissue microarrays to compare phosphorylation levels between tumor and adjacent normal tissues. Research has shown that tissues from patients with ovarian cancer are significantly more likely than adjacent normal tissues to express PEA15 phosphorylated at both S104 and S116 .

• Diabetes investigations: Given PEA15's role in glucose metabolism and its overexpression in type 2 diabetes, phospho-PEA15 (S104) antibodies can be used to examine how phosphorylation status changes in insulin-responsive tissues under diabetic conditions .

• Neurodegenerative diseases: As PEA15 is enriched in astrocytes and regulates the ERK pathway, its phosphorylation status might be relevant to neuroinflammatory processes in neurodegenerative diseases. IHC studies can reveal altered phosphorylation patterns in brain tissues .

• Phosphorylation-specific interactome: Use phospho-PEA15 (S104) antibodies for immunoprecipitation followed by mass spectrometry to identify disease-specific interaction partners that preferentially bind to the phosphorylated form.

• Therapeutic targeting: The antitumorigenic effect of non-phosphorylatable PEA15-AA suggests that inhibiting S104 phosphorylation could have therapeutic potential in certain cancers. Phospho-specific antibodies can be used to monitor the efficacy of such approaches .

What are common causes of weak or absent phospho-PEA15 (S104) signal in Western blotting?

When encountering weak or no signal:

• Rapid dephosphorylation: Phosphorylation states can be labile. Ensure samples are collected and processed rapidly with adequate phosphatase inhibitors.

• Stimulus conditions: Confirm that your experimental conditions actually induce S104 phosphorylation. Consider using positive controls like PMA-treated C6 cells .

• Antibody concentration: The recommended dilution ranges (1:500-1:2000) are starting points. Optimization may be necessary based on your specific samples and detection system .

• Blocking conditions: Excessive blocking or inappropriate blocking agents can mask epitopes. Try different blocking agents (BSA vs. non-fat dry milk) or reduce blocking time.

• Transfer efficiency: Poor transfer of small proteins like PEA15 (15 kDa) can occur. Consider using specialized transfer conditions for small proteins or PVDF membranes with smaller pore sizes.

• Detection system sensitivity: Consider switching to more sensitive detection methods, such as enhanced chemiluminescence (ECL) or fluorescent secondary antibodies.

• Antibody storage: Antibody activity may decrease with improper storage or excessive freeze-thaw cycles. Use fresh aliquots or adjust antibody concentration.

How can background issues in immunohistochemistry with phospho-PEA15 (S104) antibodies be minimized?

To reduce background in IHC applications:

• Optimal antibody dilution: Start with the recommended 1:50-1:100 dilution , but optimize for your specific tissue samples. Higher dilutions may reduce background but require longer incubation times.

• Antigen retrieval optimization: Test different antigen retrieval methods (citrate buffer, EDTA, enzymatic) and durations to find the optimal conditions for your tissue type.

• Blocking improvements: Increase blocking time or concentration, or try different blocking agents (normal serum matching the species of your secondary antibody).

• Endogenous peroxidase quenching: For HRP-based detection systems, ensure complete quenching of endogenous peroxidase activity with hydrogen peroxide.

• Endogenous biotin blocking: If using biotin-streptavidin systems, block endogenous biotin with avidin-biotin blocking kits.

• Washing optimization: Increase the number or duration of washing steps to remove unbound antibody more effectively.

• Secondary antibody cross-reactivity: Use secondary antibodies pre-adsorbed against tissues from your experimental species to reduce non-specific binding.

• Negative controls: Always include a negative control (primary antibody omitted or non-specific IgG) to assess background levels.

How can Phospho-PEA15 (S104) antibodies be integrated into single-cell analysis workflows?

For incorporating phospho-PEA15 (S104) detection into single-cell analyses:

• Phospho-flow cytometry: Adapt phospho-specific antibodies for flow cytometry by optimizing fixation and permeabilization protocols to preserve phospho-epitopes while allowing antibody access.

• Mass cytometry (CyTOF): Label phospho-PEA15 (S104) antibodies with rare earth metals for inclusion in highly multiplexed CyTOF panels to examine phosphorylation in relation to other markers at single-cell resolution.

• Single-cell Western blotting: Emerging technologies allow Western blotting on single cells; phospho-PEA15 (S104) antibodies can be adapted for these platforms to examine cell-to-cell variability in phosphorylation.

• Imaging mass cytometry: Apply metal-labeled phospho-PEA15 (S104) antibodies to tissue sections for high-dimensional spatial analysis of phosphorylation patterns at subcellular resolution.

• Spatial transcriptomics integration: Combine phospho-protein detection with spatial transcriptomics to correlate PEA15 phosphorylation status with gene expression profiles in intact tissue architecture.

What are the implications of PEA15 S104 phosphorylation for therapeutic development in cancer and diabetes?

The therapeutic relevance of PEA15 S104 phosphorylation includes:

• Cancer therapeutic targeting: The observation that non-phosphorylatable PEA15-AA has more potent antitumorigenic effects than phosphomimetic PEA15-DD suggests that inhibiting S104 phosphorylation could be therapeutically beneficial in ovarian cancer . Phospho-specific antibodies can serve as screening tools for compounds that reduce S104 phosphorylation.

• Biomarker potential: Tissue microarray studies have shown that ovarian cancer tissues are significantly more likely to express doubly-phosphorylated PEA15 compared to normal tissues . This suggests phospho-PEA15 could serve as a biomarker for certain cancers.

• Diabetes connections: PEA15 is overexpressed in type 2 diabetes and may contribute to insulin resistance in glucose uptake . Understanding how phosphorylation at S104 affects glucose metabolism could lead to novel therapeutic approaches for diabetes.

• Combination therapy approaches: Since PEA15 phosphorylation affects interaction with ERK1/2, combination therapies targeting both PEA15 phosphorylation and MAPK pathway components might have synergistic effects in cancer treatment.

• Peptide-based therapeutics: Development of peptides or small molecules that mimic non-phosphorylated PEA15 or interfere with kinases responsible for S104 phosphorylation represents a potential therapeutic strategy.

• Personalized medicine applications: Screening tumors for phosphorylated PEA15 status could help stratify patients for specific targeted therapies based on their likelihood of response.