Phospho-ELK1 (T417) Antibody

What is ELK1 and what is the significance of its phosphorylation at T417?

ELK1 is a 428 amino acid nuclear protein belonging to the Ets family of transcription factors, characterized by a highly conserved carboxy-terminal domain essential for DNA binding. This domain facilitates ELK1's interaction with specific purine-rich DNA sequences and influences transcriptional activity . The C-terminal transcriptional activation domain of ELK1 contains multiple copies of the MAPK core consensus sequence S/T-P, which can be phosphorylated by MAP kinases both in vitro and in vivo .

Phosphorylation at threonine 417 (T417) is one of several phosphorylation sites on ELK1, with others including T353, T363, T368, S383, and S389. These sites become phosphorylated with similar kinetics following serum or TPA stimulation . The phosphorylation at T417 contributes to ELK1's function as a transcriptional activator in response to mitogenic stimuli, playing a crucial role in cellular signaling pathways that regulate gene expression .

How does ELK1 T417 phosphorylation relate to the MAPK signaling pathway?

ELK1 undergoes phosphorylation by mitogen-activated protein kinase 1 (ERK) following mitogenic stimulation, which is critical for its function as a transcriptional activator . Research indicates that following ERK activation, the ELK1 C-terminal regulatory domain becomes stoichiometrically phosphorylated, receiving at least six phosphates in addition to those present prior to stimulation . The phosphorylation at T417, along with other sites, occurs following growth factor stimulation and contributes to ELK1's ability to induce target gene transcription upon JNK and MAPK-signaling pathway stimulation .

What are the recommended applications for Phospho-ELK1 (T417) antibodies?

Phospho-ELK1 (T417) antibodies are validated for several experimental applications:

• Immunohistochemistry (IHC): Particularly effective for paraffin-embedded tissues, as demonstrated in breast carcinoma samples .

• Western Blotting (WB): For detecting phosphorylated ELK1 in cell or tissue lysates .

• Immunoprecipitation (IP): For isolation and enrichment of phosphorylated ELK1 .

• Immunofluorescence (IF): For subcellular localization studies .

• In situ Proximity Ligation Assay (PLA): When used as part of an antibody pair set with antibodies against both phosphorylated and total ELK1 .

Each application requires specific optimization depending on the experimental context and sample type.

What controls should be included when using Phospho-ELK1 (T417) antibodies?

For rigorous experimental design with Phospho-ELK1 (T417) antibodies, the following controls are essential:

• Phosphopeptide Competition: Include a control where the antibody is preincubated with the synthesized phosphopeptide to confirm specificity. This approach has been demonstrated to effectively block antibody binding in immunohistochemical analysis .

• Dephosphorylation Control: Treat parallel samples with phosphatases to confirm the phospho-specificity of the antibody.

• Stimulation Controls: Include both stimulated (e.g., with serum or TPA) and unstimulated samples to demonstrate inducible phosphorylation .

• Specificity Controls: Use cells expressing mutant ELK1 where T417 has been mutated to alanine to confirm antibody specificity .

• Total ELK1 Detection: Always parallel phospho-specific detection with assessment of total ELK1 levels to normalize for expression differences.

These controls help establish confidence in experimental results and address potential non-specific binding issues.

How can I optimize immunohistochemistry protocols for Phospho-ELK1 (T417) detection?

Optimizing immunohistochemistry for Phospho-ELK1 (T417) requires careful attention to several parameters:

• Fixation and Antigen Retrieval: Phospho-epitopes can be sensitive to fixation conditions. For paraffin-embedded sections, heat-induced epitope retrieval in citrate buffer (pH 6.0) is often effective.

• Antibody Dilution: Begin with the manufacturer's recommended dilution (e.g., 1:100 as used for human breast carcinoma tissue) , then optimize through titration.

• Blocking Steps: Include thorough blocking of endogenous peroxidases and non-specific binding sites using appropriate blocking buffers.

• Incubation Conditions: Optimize both primary (Phospho-ELK1 T417) and secondary antibody incubation times and temperatures.

• Signal Development: Select an appropriate detection system based on sensitivity requirements and available equipment.

• Validation Controls: Always include positive and negative controls, including phosphopeptide-competed antibody as a specificity control .

• Preservation of Phosphorylation Status: Minimize time between tissue collection and fixation, and consider using phosphatase inhibitors during processing to prevent dephosphorylation.

What are the critical factors in sample preparation for detecting Phospho-ELK1 (T417)?

Successful detection of Phospho-ELK1 (T417) depends on preserving the phosphorylation state throughout sample preparation:

• Rapid Sample Processing: Minimize the time between sample collection and processing to prevent dephosphorylation by endogenous phosphatases.

• Phosphatase Inhibitors: Include a comprehensive phosphatase inhibitor cocktail in all lysis and processing buffers.

• Appropriate Lysis Conditions: Use lysis buffers that effectively solubilize nuclear proteins while preserving phospho-epitopes (e.g., RIPA buffer with phosphatase inhibitors).

• Temperature Control: Keep samples cold throughout processing to minimize enzymatic activity.

• Storage Considerations: Store antibodies at recommended temperatures (-20°C or lower) and avoid repeated freeze-thaw cycles by preparing small aliquots .

• Stimulation Protocols: For positive controls, stimulate cells with serum or TPA to induce ELK1 phosphorylation .

• Protein Denaturation: For applications like Western blotting, ensure complete denaturation of samples to expose the phospho-epitope.

These precautions help maintain the integrity of the phosphorylation status and improve detection sensitivity.

How does phosphorylation at T417 compare with other ELK1 phosphorylation sites?

ELK1 contains multiple phosphorylation sites including T353, T363, T368, S383, S389, and T417, all containing the MAPK core consensus sequence S/T-P . Research comparing these sites has revealed several important characteristics:

• Kinetics of Phosphorylation: Following serum or TPA stimulation, all these sites (T353, T363, T368, S383, S389, and T417) become phosphorylated with similar kinetics, suggesting coordinated regulation .

• Site Independence: Mutation experiments have demonstrated that mutation of any one site does not prevent phosphorylation of the others, indicating a degree of independence among phosphorylation events .

• Functional Significance: While phosphorylation occurs at multiple sites, mutation to alanine of S383, F378, or W379 virtually abolishes transcriptional activation by ELK1, highlighting the differential importance of specific residues .

• Stoichiometry of Phosphorylation: Two-dimensional gel electrophoresis analysis shows that following ERK activation, ELK1 receives at least six phosphates in addition to those present prior to stimulation, suggesting a complex phosphorylation pattern .

These findings highlight the complex interplay between multiple phosphorylation sites in regulating ELK1 function.

What methodological approaches can distinguish between different phosphorylated forms of ELK1?

Distinguishing between specific phosphorylation sites on ELK1 requires specialized techniques:

These complementary approaches provide a comprehensive understanding of the complex phosphorylation patterns of ELK1.

How can I integrate Phospho-ELK1 (T417) data with transcriptomic analyses?

Integrating phosphorylation data with transcriptomics requires sophisticated experimental design and analysis:

• Temporal Coordination: Design experiments that capture both phosphorylation dynamics and subsequent transcriptional changes with appropriate time points.

• Cell Systems: Use well-characterized cell systems where ELK1 target genes are known, such as those expressing immediate early genes like FOS and IER2 .

• Perturbation Approaches: Combine specific pathway activators/inhibitors with phospho-ELK1 detection and RNA-seq to establish causality.

• Chromatin Immunoprecipitation (ChIP-seq): Use phospho-specific ELK1 antibodies in ChIP-seq to identify genomic binding sites of phosphorylated ELK1.

• Integrated Bioinformatics Pipeline:

  • Normalize phosphorylation signals to total protein levels

  • Correlate phosphorylation intensity with expression changes of potential target genes

  • Perform pathway enrichment analysis of differentially expressed genes

  • Integrate with known ELK1 binding motifs from ChIP-seq data

• Validation Experiments: Use reporter gene assays with wild-type and phospho-mutant ELK1 to confirm the functional significance of T417 phosphorylation on specific target genes.

This integrated approach helps establish the mechanistic link between T417 phosphorylation and transcriptional outcomes.

How should I interpret contradictory results in ELK1 phosphorylation studies?

When faced with contradictory results in ELK1 phosphorylation studies, consider these methodological and biological factors:

• Antibody Specificity: Different phospho-specific antibodies may have varying degrees of specificity and cross-reactivity with other phosphorylation sites. Verify antibody specificity through peptide competition and mutagenesis experiments .

• Temporal Dynamics: Phosphorylation is dynamic, and discrepancies may result from differences in sampling times. Design time-course experiments to capture the complete phosphorylation profile.

• Cell Type Variations: Different cell types may exhibit different signaling dynamics or express varying levels of phosphatases. Compare results across multiple cell types and relate to biological context.

• Stimulation Conditions: The strength, duration, and type of stimulus affect phosphorylation patterns. Standardize stimulation protocols and include positive controls (e.g., serum or TPA stimulation) .

• Technical Variations: Different detection methods (Western blot, immunohistochemistry, mass spectrometry) have different sensitivities and specificities. Validate findings using complementary techniques.

• Normalization Approaches: Ensure proper normalization to total ELK1 protein levels when quantifying phosphorylation.

• Statistical Analysis: Apply appropriate statistical tests and consider biological versus technical replicates when interpreting significance.

When reporting contradictory results, clearly document all methodological details to facilitate interpretation and future replication.

What are common pitfalls in Phospho-ELK1 (T417) experimental design and how can they be avoided?

Several common pitfalls can compromise Phospho-ELK1 (T417) experiments:

• Insufficient Phosphorylation Preservation:

  • Pitfall: Loss of phosphorylation during sample processing

  • Solution: Use phosphatase inhibitors consistently throughout all procedures and process samples rapidly at cold temperatures

• Inadequate Controls:

  • Pitfall: Inability to distinguish specific from non-specific signals

  • Solution: Include phosphopeptide competition controls, phosphatase-treated samples, and mutant cell lines (T417A)

• Cross-reactivity Issues:

  • Pitfall: Antibodies detecting other phosphorylated sites

  • Solution: Validate specificity through peptide competition and mutagenesis experiments

• Non-physiological Conditions:

  • Pitfall: Studying phosphorylation under artificial conditions

  • Solution: Use physiologically relevant stimulation conditions and validate findings in multiple models

• Inappropriate Time Points:

  • Pitfall: Missing dynamic changes in phosphorylation

  • Solution: Include comprehensive time courses following stimulation

• Storage and Handling Issues:

  • Pitfall: Antibody degradation affecting performance

  • Solution: Store antibodies at recommended temperatures (-20°C or lower) and avoid repeated freeze-thaw cycles by preparing small aliquots

• Inadequate Quantification:

  • Pitfall: Relying on visual assessment of signal intensity

  • Solution: Use appropriate image analysis software and normalize to loading controls

Careful experimental design that addresses these potential pitfalls will improve data quality and interpretability.

How does ELK1 T417 phosphorylation relate to SUMOylation and transcriptional regulation?

The relationship between ELK1 phosphorylation and SUMOylation represents an important regulatory mechanism:

• Antagonistic Relationship: Phosphorylation of ELK1 following mitogenic stimulation leads to SUMOylation release, a post-translational modification that otherwise recruits histone deacetylase 2 (HDAC2) to target gene promoters .

• Histone Modification Effects: When SUMOylation is present, it leads to decreased histone acetylation and diminished transactivator activity of ELK1. Phosphorylation, including at T417, counteracts this effect .

• Signaling Integration: This phosphorylation-SUMOylation switch allows ELK1 to integrate multiple signaling inputs and fine-tune transcriptional responses.

• Temporal Regulation: The kinetics of phosphorylation at T417 and other sites, along with subsequent SUMOylation changes, determine the duration and intensity of ELK1-mediated transcriptional activation.

• Target Gene Specificity: The interplay between phosphorylation and SUMOylation may contribute to selectivity in ELK1 target gene activation, with different targets showing different sensitivities to this regulatory mechanism.

This complex interplay highlights how post-translational modifications work in concert to regulate transcription factor activity.

What is the role of ELK1 T417 phosphorylation in pathological conditions?

Emerging research points to several potential roles for ELK1 T417 phosphorylation in disease contexts:

• Cancer Biology: Phosphorylated ELK1 has been detected in breast carcinoma tissues using immunohistochemistry, suggesting potential involvement in cancer progression . The role of ELK1 in regulating immediate early genes like FOS connects it to cell proliferation pathways often dysregulated in cancer.

• Neurological Disorders: As a transcription factor responsive to MAPK signaling, phosphorylated ELK1 may play a role in neuronal plasticity and survival, with potential implications for neurodegenerative diseases.

• Inflammatory Responses: The MAPK pathways that phosphorylate ELK1 are often activated during inflammation, suggesting a potential role for ELK1 phosphorylation in inflammatory disorders.

• Therapeutic Targeting: Understanding the specific contribution of T417 phosphorylation to ELK1 function in disease contexts could reveal new therapeutic approaches targeting either the phosphorylation event itself or downstream effects.

• Biomarker Potential: The detection of phosphorylated ELK1 in patient samples might serve as a biomarker for pathway activation in certain diseases, potentially guiding treatment decisions.

Further research using phospho-specific antibodies in patient-derived samples and disease models will help clarify these potential roles.

What novel methodologies are being developed to study ELK1 phosphorylation dynamics in living cells?

Several cutting-edge approaches are advancing our understanding of ELK1 phosphorylation dynamics:

• Phospho-specific FRET Sensors: Genetically encoded biosensors that report on ELK1 phosphorylation state in real-time in living cells through fluorescence resonance energy transfer.

• Optogenetic Approaches: Light-controlled activation of signaling pathways combined with phospho-specific detection to achieve precise temporal control over ELK1 phosphorylation.

• Live-Cell Imaging with Antibody Fragments: Cell-permeable antibody fragments or nanobodies specific to phosphorylated ELK1 enabling real-time visualization of phosphorylation events.

• Single-Cell Phospho-Proteomics: Emerging technologies that can measure phosphorylation events in individual cells, revealing heterogeneity in ELK1 phosphorylation patterns within populations.

• Proximity Ligation Assay Adaptations: In situ PLA techniques using antibody pairs against both phosphorylated and total ELK1 provide spatial information about phosphorylation events .

• CRISPR-Cas9 Engineered Reporter Systems: Endogenous tagging of ELK1 combined with phospho-sensors to monitor native protein modifications without overexpression artifacts.

These advanced methodologies promise to provide unprecedented insights into the spatial and temporal dynamics of ELK1 phosphorylation in physiologically relevant contexts.

What are the recommended storage and handling procedures for Phospho-ELK1 (T417) antibodies?

To maintain antibody performance and longevity, follow these storage and handling recommendations:

• Temperature Conditions:

  • Store antibodies at -20°C or lower for long-term storage

  • Avoid storing at 4°C for extended periods

  • Prepare working aliquots to avoid repeated freeze-thaw cycles

• Buffer Composition:

  • Many phospho-specific antibodies are supplied in PBS with 0.02% sodium azide and 50% glycerol at pH 7.2-7.4

  • Do not alter buffer composition unless specifically recommended

• Aliquoting Protocol:

  • Thaw antibody completely at 4°C before aliquoting

  • Use sterile tubes and pipette tips

  • Prepare small single-use aliquots (5-20 μl) to minimize freeze-thaw cycles

  • Return aliquots to -20°C storage immediately after use

• Handling During Experiments:

  • Keep on ice when in use

  • Return to appropriate storage promptly

  • Avoid contamination with microorganisms

• Expiration Considerations:

  • Document date of receipt and initial use

  • Track number of freeze-thaw cycles

  • Validate antibody performance periodically, especially after extended storage

Proper storage and handling will maximize antibody stability and experimental reproducibility.

What is the recommended protocol for validating a new lot of Phospho-ELK1 (T417) antibody?

When validating a new antibody lot, implement this comprehensive validation protocol:

• Positive Control Samples:

  • Use cells stimulated with serum or TPA to induce ELK1 phosphorylation

  • Include samples with known positive signal from previous antibody lots

• Specificity Validation:

  • Perform phosphopeptide competition assays using the synthesized phosphopeptide derived from human ELK1 around T417

  • Include dephosphorylated samples (phosphatase-treated) as negative controls

  • Test on samples expressing mutant ELK1 (T417A) if available

• Application-specific Validation:

  • For Western blotting: Verify molecular weight and band pattern

  • For IHC: Compare staining pattern with published results and previous lot performance in positive control tissues

  • For IF: Evaluate subcellular localization pattern

• Cross-reactivity Assessment:

  • Test on multiple relevant species if cross-reactivity is claimed

  • Check for non-specific binding in knockout or knockdown samples if available

• Titration Experiments:

  • Test multiple antibody dilutions to determine optimal working concentration

  • Compare sensitivity and signal-to-noise ratio with previous lot

• Lot-to-lot Comparison:

  • Run side-by-side experiments with the previous lot on identical samples

  • Document any differences in sensitivity, specificity, or background

• Documentation:

  • Maintain detailed records of all validation experiments

  • Archive validation images for future reference

This systematic approach ensures experimental continuity and reliable results when transitioning to a new antibody lot.

How can Phospho-ELK1 (T417) antibodies be applied in cancer research?

Phospho-ELK1 (T417) antibodies offer valuable tools for investigating cancer biology:

• Diagnostic Applications:

  • Immunohistochemical analysis of tumor tissues to assess MAPK pathway activation status

  • Evaluation of phospho-ELK1 levels in breast carcinoma and other cancer types

• Signaling Pathway Analysis:

  • Monitoring treatment response to MAPK pathway inhibitors in cancer cells

  • Studying resistance mechanisms to targeted therapies

• Biomarker Development:

  • Correlation of ELK1 phosphorylation patterns with clinical outcomes

  • Potential stratification marker for selecting patients likely to respond to specific treatments

• Functional Studies:

  • Investigation of ELK1's role in regulating cancer-relevant immediate early genes

  • Assessment of how phosphorylation at T417 affects oncogenic transcriptional programs

• Drug Discovery Applications:

  • Screening compounds that modulate ELK1 phosphorylation

  • Evaluating on-target effects of kinase inhibitors targeting the MAPK pathway

• Combination Therapy Rationales:

  • Understanding how modulation of ELK1 phosphorylation might enhance effectiveness of other cancer therapies

These applications highlight the potential of phospho-ELK1 antibodies as important tools in translational cancer research.

What are the cutting-edge research questions about ELK1 T417 phosphorylation?

Several frontier research questions are driving current investigations into ELK1 T417 phosphorylation:

• Phosphorylation Code Deciphering:

  • How does the pattern of phosphorylation across multiple sites (including T417) encode specific transcriptional outputs?

  • Are there preferred sequences or hierarchies of phosphorylation events?

• Cross-talk with Other Modifications:

  • How does T417 phosphorylation interact with other post-translational modifications like SUMOylation and acetylation?

  • What is the mechanistic basis for the antagonism between phosphorylation and SUMOylation ?

• Single-Cell Heterogeneity:

  • How variable is ELK1 T417 phosphorylation at the single-cell level?

  • Does heterogeneity in ELK1 phosphorylation contribute to cell fate decisions?

• Non-transcriptional Functions:

  • Does phosphorylated ELK1 have functions beyond transcriptional regulation?

  • Are there cytoplasmic roles for phosphorylated ELK1?

• Therapeutic Targeting:

  • Can ELK1 phosphorylation be selectively modulated for therapeutic benefit?

  • What are the downstream consequences of inhibiting specific phosphorylation events?

• Structural Biology Questions:

  • How does phosphorylation at T417 affect the three-dimensional structure of ELK1?

  • What are the conformational changes induced by multiple phosphorylation events?

These questions represent fertile ground for researchers using phospho-specific antibodies and other advanced tools to understand ELK1 regulation.