Phospho-ELK1 (S389) Antibody

What is ELK1 and what role does phosphorylation at Ser389 play in its function?

ELK1 functions as a critical component of the ternary complex that binds to the serum response element (SRE) and mediates gene activity in response to serum and growth factors. The protein undergoes phosphorylation by MAP kinase pathways at multiple S/T motifs located at its C-terminus, with phosphorylation at Ser389 being particularly significant for its activity . This post-translational modification is crucial for transcriptional activation of ELK1 and represents a key regulatory step in the signaling cascade .

ELK1 serves as a nuclear target for the ras-raf-MAPK signaling cascade, with phosphorylation events at sites like Ser389 directly linking cytoplasmic signaling to nuclear gene regulation . While Ser383 phosphorylation has received significant attention in earlier studies, research indicates that phosphorylation at Ser389 works in concert with Ser383 modification to achieve maximal activation of ELK1-mediated transcription .

How does phosphorylation at Ser389 affect ELK1 structure and function?

Phosphorylation at Ser389 induces significant conformational changes in both the secondary and tertiary structure of ELK1. Spectroscopic studies using circular dichroism (CD) and fluorescence emission have demonstrated that phosphorylation events, including at Ser389, increase α-helicity in the protein structure and alter the tertiary conformation . These structural changes directly influence:

• DNA binding capacity - Phosphorylation increases ELK1's affinity for target DNA sequences

• Protein-protein interactions - Although phosphorylation does not appear to significantly alter binding efficiency with SRF as shown by GST pull-down assays

• Transcriptional activation potential - The conformational shift exposes functional domains necessary for recruiting transcriptional machinery

The structural change appears to involve interactions between the phosphorylated C-terminal activation domain and the ETS domain, as evidenced by fluorescence emission spectroscopy showing altered environments around tryptophan residues following phosphorylation .

How can phospho-specific ELK1 (S389) antibodies be used to study signal transduction dynamics?

Phospho-specific antibodies against ELK1 (S389) provide powerful tools for studying signaling kinetics in real-time. When designing experiments to investigate signal transduction dynamics, researchers should consider:

• Time-course experiments: Monitoring phosphorylation at multiple time points (0-60 minutes) following stimulation can reveal the temporal dynamics of ELK1 activation. Evidence suggests maximum phosphorylation occurs after approximately 60 minutes of stimulation, coinciding with maximal DNA binding capacity .

• Spatial distribution analysis: Combining phospho-ELK1 (S389) antibodies with subcellular fractionation or immunofluorescence microscopy enables tracking of the activated transcription factor's translocation between cytoplasmic and nuclear compartments.

• Multiplexed pathway analysis: Using phospho-ELK1 (S389) antibodies alongside markers for upstream kinases (like ERK, JNK, or p38) allows for comprehensive pathway mapping and identification of rate-limiting steps in the signaling cascade.

• Cell-based ELISAs: These provide quantitative measurement of phosphorylated ELK1 levels in response to different stimuli, allowing for high-throughput screening of pathway modulators .

For optimal results, normalize phospho-ELK1 (S389) levels to total ELK1 protein expression to account for variations in baseline protein abundance across experimental conditions.

What is the relationship between multi-site phosphorylation of ELK1 and its functional outcomes?

• Threshold effects: High levels of phosphorylation are required to achieve maximal DNA binding capacity, suggesting a cooperative mechanism between phosphorylation sites .

• Temporal coordination: Different phosphorylation sites may be modified in a specific sequence, with early phosphorylation events priming the protein for subsequent modifications.

• Conformational heterogeneity: Mutational studies involving Elk-1(S383A/S389A) demonstrate that these mutants can still undergo phosphorylation-induced conformational changes, albeit defective ones, indicating that multiple determinants influence the precise structural changes induced by phosphorylation .

• Functional specialization: Different phosphorylation patterns may direct ELK1 toward specific gene targets or protein interaction partners.

When investigating multi-site phosphorylation, researchers should employ phospho-specific antibodies against various sites in combination with mass spectrometry to map the complete phosphorylation landscape and correlate specific patterns with downstream outcomes.

What are the optimal conditions for using Phospho-ELK1 (S389) antibodies in Western blot applications?

For successful Western blot detection of phosphorylated ELK1 at Ser389, researchers should follow these methodological guidelines:

• Sample preparation:

  • Rapidly harvest and lyse cells to prevent dephosphorylation by endogenous phosphatases

  • Include phosphatase inhibitors (e.g., sodium fluoride, sodium orthovanadate) in lysis buffers

  • Maintain samples at 4°C throughout processing

• Gel electrophoresis and transfer:

  • Use 10-12% polyacrylamide gels to achieve optimal resolution of ELK1 (approximately 45 kDa)

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

  • Use methanol-free transfer buffers to enhance transfer efficiency of phosphorylated proteins

• Antibody incubation:

  • Block membranes with 5% BSA in TBST (not milk, which contains phospho-proteins)

  • Dilute primary phospho-ELK1 (S389) antibody at 1:1000 in blocking buffer

  • Incubate overnight at 4°C with gentle agitation

  • Use HRP-conjugated anti-rabbit secondary antibody at 1:2000-1:5000 dilution

• Detection and validation:

  • Include both phosphorylated and non-phosphorylated control samples

  • Consider using lambda phosphatase-treated samples as negative controls

  • Compare results with total ELK1 antibody staining on parallel blots or after stripping

Expected results include detection of a band at approximately 45 kDa that increases in intensity following stimulation with growth factors or activators of the MAPK pathway .

How can Phospho-ELK1 (S389) antibodies be optimized for immunohistochemistry applications?

Optimizing immunohistochemistry (IHC) protocols for phospho-ELK1 (S389) detection requires careful attention to tissue preservation, antigen retrieval, and signal amplification:

• Tissue preparation:

  • Use freshly fixed tissues or properly stored paraffin-embedded specimens

  • Perfusion fixation is preferable for animal tissues to preserve phospho-epitopes

  • Limit fixation time to prevent masking of phospho-epitopes

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Optimize retrieval time (10-20 minutes) to balance epitope exposure and tissue integrity

  • Allow slides to cool slowly to room temperature after retrieval

• Antibody application:

  • Apply phospho-ELK1 (S389) antibody at 1:50-1:100 dilution

  • Incubate overnight at 4°C in a humidified chamber

  • Use signal amplification systems (e.g., ABC, polymer detection) for enhanced sensitivity

• Controls and validation:

  • Include phosphatase-treated sections as negative controls

  • Use activator-treated and untreated samples for comparison

  • Perform peptide competition assays using the phosphopeptide immunogen to confirm specificity

Successful IHC staining typically shows nuclear localization of phospho-ELK1 (S389), with intensity varying based on tissue type and activation state of the MAPK pathway.

How should experiments be designed to study the kinetics of ELK1 phosphorylation at Ser389?

Designing experiments to study ELK1 phosphorylation kinetics requires careful consideration of temporal resolution, stimulus parameters, and detection methods:

• Stimulus selection and optimization:

  • Choose physiologically relevant stimuli (e.g., growth factors, serum, stress inducers)

  • Determine optimal stimulus concentration through dose-response experiments

  • Consider using specific pathway activators (e.g., phorbol esters for PKC/MAPK) alongside broader stimuli

• Time-course design:

  • Include early time points (30 seconds, 2, 5, 10 minutes) to capture rapid phosphorylation

  • Extend observations to 60+ minutes to monitor sustained phosphorylation events

  • Evidence suggests maximal ELK1 phosphorylation occurs around 60 minutes post-stimulation

• Detection methods:

  • Western blotting with phospho-ELK1 (S389) antibodies for semi-quantitative analysis

  • Cell-based ELISAs for higher-throughput quantification

  • Phosphoproteomics for comprehensive multi-site phosphorylation analysis

• Data analysis approach:

  • Plot phosphorylation intensity versus time

  • Calculate rate constants for phosphorylation and dephosphorylation phases

  • Correlate phosphorylation kinetics with downstream functional outcomes (e.g., gene expression)

For meaningful interpretation, always normalize phospho-ELK1 (S389) signal to total ELK1 levels to account for variations in protein expression.

What are common pitfalls when detecting phospho-ELK1 (S389) and how can they be addressed?

Researchers frequently encounter several challenges when working with phospho-ELK1 (S389) antibodies that can be addressed through methodological refinements:

• Low signal intensity:

  • Cause: Rapid dephosphorylation during sample preparation

  • Solution: Immediately lyse samples in buffers containing phosphatase inhibitors; maintain samples at 4°C throughout processing

• High background:

  • Cause: Non-specific antibody binding or inadequate blocking

  • Solution: Use 5% BSA instead of milk for blocking; optimize antibody dilutions; increase washing times and volumes

• Inconsistent results between replicates:

  • Cause: Variable baseline phosphorylation or technical variability

  • Solution: Synchronize cells prior to experiments; standardize cell densities and lysis conditions; include internal control samples across experiments

• Cross-reactivity with other phospho-proteins:

  • Cause: Antibody recognizing similar phospho-epitopes on other proteins

  • Solution: Validate specificity using knockout/knockdown samples or peptide competition assays; compare results with alternative phospho-ELK1 antibodies

• Inability to detect phosphorylation changes:

  • Cause: Suboptimal stimulation conditions or timing

  • Solution: Verify pathway activation using established markers (e.g., phospho-ERK); optimize stimulus concentration and exposure time; consider cell type-specific response kinetics

When troubleshooting, systematically modify one variable at a time while maintaining appropriate controls to identify the specific source of the issue.

How does the phosphorylation status of ELK1 at Ser389 correlate with conformational changes and DNA binding activity?

The relationship between ELK1 phosphorylation at Ser389 and its functional properties has been characterized through multiple experimental approaches that reveal a mechanistic connection:

• Conformational dynamics:

  • Phosphorylation at Ser389, along with other sites, induces measurable changes in both secondary and tertiary structure of ELK1

  • Circular dichroism studies show increased α-helicity following phosphorylation

  • Fluorescence emission spectroscopy demonstrates altered environments around tryptophan residues, indicating tertiary structural rearrangements

• Structure-function relationship:

  • The B-box domain plays a critical role in mediating phosphorylation-induced conformational changes

  • Mutational analysis (e.g., L158P mutation) reveals that while secondary structural changes still occur, tertiary structural alterations are compromised in this mutant

  • This structure-function relationship directly impacts DNA binding capacity

• DNA binding capacity correlation:

  • Maximum DNA binding is achieved at high levels of phosphorylation (around 60 minutes post-stimulation)

  • The stoichiometry of phosphorylation is crucial, suggesting cooperative effects between multiple phosphorylation sites

  • Phosphorylation releases an inhibitory intramolecular interaction, allowing the ETS domain to effectively engage with DNA

• Protein-protein interactions:

  • Interestingly, while phosphorylation enhances DNA binding, GST pull-down assays indicate that interaction with SRF is not significantly affected by phosphorylation status

  • This suggests that phosphorylation primarily regulates ELK1 function through conformational effects rather than by directly modulating protein partner selection

These insights provide a molecular framework for understanding how phosphorylation events translate into functional outputs in transcriptional regulation mediated by ELK1.

How can cell-based ELISA approaches using phospho-ELK1 (S389) antibodies enhance signaling pathway analysis?

Cell-based ELISA methodologies offer unique advantages for studying phospho-ELK1 (S389) in intact cellular contexts:

• Quantitative capabilities:

  • Cell-based ELISAs provide precise quantification of phosphorylation levels in response to different stimuli

  • Results can be normalized to total cell number using crystal violet staining, enabling adjustment for plating differences

  • This approach allows for direct comparison of phosphorylation levels across different experimental conditions

• High-throughput applications:

  • The 96-well microplate format enables testing of multiple conditions simultaneously

  • This is particularly valuable for screening pathway modulators, inhibitor dose-response studies, or time-course analyses

  • The scalable nature conserves cell culture and treatment reagents compared to Western blotting

• Experimental workflow:

  • Cells are cultured directly in 96-well plates

  • Following stimulation, cells are fixed and permeabilized

  • Detection uses target-specific primary antibodies against phospho-ELK1 (S389) followed by HRP-conjugated secondary antibodies

  • Colorimetric measurement of HRP activity provides quantitative readout of phosphorylation levels

• Multiplexed analysis potential:

  • Parallel wells can be probed for multiple signaling components

  • This allows for comprehensive pathway mapping by simultaneously monitoring phosphorylation of ELK1 alongside upstream kinases and downstream effectors

  • Relative activation levels of different pathway components can reveal bottlenecks or critical nodes in signal transduction

When implementing cell-based ELISAs, researchers should optimize cell density, fixation conditions, and antibody concentrations to ensure maximum sensitivity and specificity for phospho-ELK1 (S389) detection.

How can phospho-ELK1 (S389) analysis be integrated with gene expression studies to understand transcriptional regulation?

Integrating phospho-ELK1 (S389) analysis with transcriptomic approaches provides a comprehensive view of signal-dependent gene regulation:

• Temporal correlation analyses:

  • Monitor phospho-ELK1 (S389) levels alongside expression of known ELK1 target genes

  • Establish time-lag relationships between peak phosphorylation and maximum transcriptional output

  • This temporal mapping can reveal the kinetic relationship between signaling events and gene expression changes

• ChIP-seq integration:

  • Combine phospho-ELK1 (S389) quantification with chromatin immunoprecipitation sequencing

  • Determine whether phosphorylation status correlates with genomic occupancy patterns

  • Identify consensus binding motifs for phosphorylated versus non-phosphorylated ELK1

• Multi-omics experimental design:

  • Collect matched samples for phospho-protein analysis and RNA-seq from the same experimental conditions

  • Use statistical approaches to correlate phosphorylation dynamics with transcriptional profiles

  • Employ network analysis to identify phospho-ELK1 (S389)-dependent gene modules

• Functional validation strategies:

  • Express phosphomimetic (S389D/E) or phospho-deficient (S389A) ELK1 mutants

  • Compare resulting gene expression profiles to identify phosphorylation-dependent transcriptional programs

  • Validate key regulatory relationships using reporter assays or CRISPR-based approaches

This integrated approach allows researchers to move beyond correlative observations to establish causal relationships between ELK1 phosphorylation states and specific transcriptional outcomes.

What techniques can be used to study the interaction between phosphorylated ELK1 and chromatin remodeling complexes?

Investigating interactions between phospho-ELK1 (S389) and chromatin regulators requires specialized approaches:

• Co-immunoprecipitation strategies:

  • Immunoprecipitate phospho-ELK1 (S389) from nuclear extracts using phospho-specific antibodies

  • Identify associated chromatin modifiers through immunoblotting or mass spectrometry

  • Compare interaction profiles between phosphorylated and non-phosphorylated states

  • Consider crosslinking approaches to capture transient interactions

• Proximity ligation assays:

  • Visualize in situ interactions between phospho-ELK1 (S389) and chromatin regulators

  • Quantify interaction dynamics following pathway activation

  • This technique is particularly valuable for detecting interactions in their native chromatin context

• ChIP-sequential approaches:

  • Perform sequential ChIP first with phospho-ELK1 (S389) antibodies followed by antibodies against chromatin modifiers

  • Identify genomic loci where both proteins co-occupy

  • This technique can reveal the subset of ELK1 target genes that undergo specific chromatin modifications

• FRET-based interaction analysis:

  • Generate fluorescently tagged ELK1 with phosphomimetic mutations

  • Measure energy transfer between tagged ELK1 and chromatin regulators

  • This approach can provide kinetic information about complex formation and dissociation

The phosphorylation-induced conformational change in ELK1 likely creates binding surfaces for specific chromatin regulators, representing a direct mechanism linking signaling events to epigenetic modulation at target genes.

What are emerging technologies for studying phospho-ELK1 (S389) dynamics in living cells?

Cutting-edge approaches for real-time monitoring of phospho-ELK1 (S389) in living systems include:

• Phospho-specific biosensors:

  • Engineered FRET-based sensors incorporating ELK1 domains and phospho-binding modules

  • These sensors undergo conformational changes upon phosphorylation, generating measurable signals

  • Enable real-time visualization of phosphorylation events in single cells with high temporal resolution

• Proximity-based labeling approaches:

  • TurboID or APEX2 fusion proteins to identify the dynamic interactome of phosphorylated ELK1

  • Temporal control allows mapping of interaction partners at different stages of phosphorylation

  • This technique can reveal how phosphorylation at Ser389 reconfigures the molecular environment of ELK1

• Live-cell phospho-protein imaging:

  • Antibody-based detection using cell-permeable nanobodies against phospho-ELK1 (S389)

  • Genetically encoded phospho-specific intrabodies that selectively recognize phosphorylated epitopes

  • These approaches enable tracking of endogenous phospho-ELK1 (S389) in living cells without overexpression artifacts

• Optogenetic control of ELK1 phosphorylation:

  • Light-activatable kinases or phosphatases targeting ELK1

  • Enables precise spatiotemporal control of phosphorylation status

  • Allows direct testing of the functional consequences of Ser389 phosphorylation with minimal pathway cross-activation

These emerging technologies promise to overcome limitations of traditional biochemical approaches by providing dynamic, spatially resolved information about phosphorylation events within their native cellular context.

How might single-cell analysis of phospho-ELK1 (S389) advance our understanding of cellular heterogeneity in signaling responses?

Single-cell approaches to studying phospho-ELK1 (S389) can reveal important insights about signaling heterogeneity:

• Mass cytometry (CyTOF) applications:

  • Metal-conjugated antibodies against phospho-ELK1 (S389) enable quantification at the single-cell level

  • Simultaneous measurement of multiple phospho-proteins reveals correlations between different pathway components

  • Clustering approaches can identify distinct cell states based on phosphorylation profiles

  • This technique is particularly valuable for analyzing heterogeneous tissues or mixed cell populations

• Single-cell phospho-proteomics:

  • Emerging techniques for phospho-protein analysis at the single-cell level

  • Can reveal cell-to-cell variability in ELK1 phosphorylation patterns

  • Potential to identify rare cell populations with unique phosphorylation signatures

• Spatial considerations:

  • Imaging mass cytometry or multiplexed immunofluorescence to map phospho-ELK1 (S389) distribution

  • Analysis of spatial relationships between phospho-ELK1 positive cells and tissue microenvironments

  • This approach can reveal how local signaling niches influence ELK1 phosphorylation states

• Integrated multi-modal analysis:

  • Combine single-cell transcriptomics with phospho-protein detection

  • Establish direct links between phosphorylation status and gene expression programs at the single-cell level

  • This integrated view can elucidate how signaling heterogeneity translates to transcriptional diversity

Understanding cellular heterogeneity in phospho-ELK1 (S389) responses could reveal new insights into differential cellular responses to the same stimulus and identify subpopulations with unique signaling behaviors relevant to development or disease.