Phospho-ELK1 (S383) Antibody

What is ELK1 and what is the significance of its phosphorylation at serine 383?

ELK1 is a transcription factor belonging to the E-twenty-six (ETS) domain superfamily that binds to purine-rich DNA sequences. It forms a ternary complex with Serum Response Factor (SRF) and the ETS and SRF motifs of the serum response element (SRE) on promoter regions of immediate early genes such as FOS and IER2. Phosphorylation at serine 383 is critical for ELK1 activation and occurs primarily through the ERK/MAPK pathway. This phosphorylation event is a key molecular switch that induces target gene transcription upon JNK and MAPK-signaling pathway stimulation. The phosphorylation status of S383 serves as an important indicator of transcriptional activity of ELK1 and reflects upstream MAPK pathway activation.

How does the specificity of Phospho-ELK1 (S383) antibodies compare to pan-ELK1 antibodies?

Phospho-ELK1 (S383) antibodies specifically recognize the phosphorylated form of ELK1 at serine 383, making them ideal for monitoring MAPK-dependent activation of this transcription factor. In contrast, pan-ELK1 antibodies detect total ELK1 protein regardless of its phosphorylation status. Validation studies consistently show that phospho-specific antibodies only detect ELK1 protein in stimulated conditions (such as after UV treatment or MAPK pathway activation), while showing minimal to no reactivity with unphosphorylated ELK1. Western blot analysis confirms this specificity, as demonstrated by the selective immunolabeling of the ~46 kDa phosphorylated ELK1 protein band compared to unphosphorylated controls. Additionally, the specificity can be verified through blocking experiments with phospho-peptides, which eliminate the signal in both Western blot and immunohistochemistry applications.

What is the molecular weight of phosphorylated ELK1, and how can proper detection be confirmed?

The predicted molecular weight of ELK1 is approximately 45-46 kDa. In Western blot applications, researchers should expect to observe a band at this position when using phospho-ELK1 (S383) antibodies on appropriately stimulated samples. Proper detection can be confirmed through several validation approaches:

• Parallel running of stimulated samples (e.g., UV-treated or growth factor-stimulated cells) alongside unstimulated controls.

• Inclusion of phosphatase-treated lysates as negative controls.

• Phospho-peptide competition assays, where pre-incubation of the antibody with the phosphopeptide immunogen should abolish or significantly reduce the signal.

• Molecular weight verification using protein ladders to ensure the detected band corresponds to the expected 45-46 kDa size.

What are the optimal protocols for Western blot detection of Phospho-ELK1 (S383)?

For optimal Western blot detection of Phospho-ELK1 (S383), the following methodological considerations are recommended:

• Sample preparation: Cells should be lysed in buffers containing phosphatase inhibitors to preserve the phosphorylation status. Flash-freezing samples immediately after collection is advisable.

• Antibody dilution: The recommended working dilution ranges from 1:500 to 1:2000, though optimal concentrations should be determined empirically for each experimental setup.

• Blocking conditions: Use 5% BSA in TBST rather than milk-based blockers, as milk contains phosphatases that may reduce phospho-specific signals.

• Detection method: Enhanced chemiluminescence (ECL) technique has been validated for visualization, with exposure times optimized based on signal intensity.

• Controls: Include both phosphorylated (e.g., UV-treated HeLa cells) and unphosphorylated recombinant ELK1 protein as positive and negative controls, respectively.

• Stripping and reprobing: If assessing total ELK1 on the same membrane, mild stripping conditions are recommended to preserve epitope integrity.

How should Phospho-ELK1 (S383) antibody be optimized for immunohistochemistry applications?

For immunohistochemistry applications using Phospho-ELK1 (S383) antibody, the following optimization steps are crucial:

• Tissue fixation and processing: Formalin-fixed, paraffin-embedded (FFPE) tissues have been validated with this antibody. Optimal fixation time should be determined to balance antigen preservation and tissue morphology.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is typically effective. The optimal method should be determined empirically.

• Antibody dilution: Start with dilutions between 1:100 and 1:300, and adjust based on signal-to-noise ratio.

• Incubation conditions: Overnight incubation at 4°C often yields optimal results for phospho-specific antibodies.

• Detection system: Use detection systems appropriate for rabbit IgG primaries, with careful optimization of the amplification step to avoid background.

• Controls: Include phospho-peptide blocking controls to demonstrate specificity; the signal should be significantly reduced or eliminated when the antibody is pre-incubated with the phospho-peptide.

What experimental conditions trigger ELK1 S383 phosphorylation for positive control preparation?

To prepare robust positive controls for Phospho-ELK1 (S383) detection, several experimental conditions that trigger ELK1 phosphorylation can be employed:

• Serum stimulation: Serum-starved cells (0.5-1% serum for 16-24 hours) followed by stimulation with 10-20% serum for 15-30 minutes effectively activates the MAPK pathway and induces S383 phosphorylation.

• Growth factor treatment: EGF (50-100 ng/ml, 5-15 minutes), PDGF, or FGF treatment rapidly activates the ERK pathway leading to ELK1 phosphorylation.

• Phorbol esters: Treatment with PMA (100 nM, 30 minutes) stimulates PKC and downstream MAPK activation.

• UV irradiation: Short exposure to UV light (e.g., 40 J/m² of UVC) effectively induces stress-activated MAPK pathways leading to ELK1 phosphorylation.

• Pharmacological activators: MEK activators or phosphatase inhibitors (such as okadaic acid) can enhance phosphorylation levels.

Cell collection should occur at the peak of phosphorylation (typically 15-30 minutes after stimulation) and samples should be immediately processed in phosphatase inhibitor-containing buffers to preserve the phosphorylation signal.

How does Phospho-ELK1 (S383) localization change during the cell cycle, and what methods best capture these dynamics?

Phospho-ELK1 (S383) exhibits distinct localization patterns throughout the cell cycle, with significant implications for understanding its non-transcriptional functions. Research has revealed that S383-phosphorylated ELK1 associates with mitotic spindle poles from metaphase through telophase and relocates to the spindle midbody during cytokinesis, suggesting a potential role in cell division regulation beyond its established transcriptional function.

To effectively study these dynamics, researchers should consider:

• Synchronized cell populations: Using double thymidine block or nocodazole treatment followed by release to enrich for specific cell cycle phases.

• Co-immunofluorescence approaches: Combining Phospho-ELK1 (S383) antibody with markers for mitotic structures (e.g., α-tubulin for spindles, γ-tubulin for centrosomes) and DNA staining to precisely map localization during mitotic progression.

• Live-cell imaging: For dynamic studies, expression of fluorescently-tagged ELK1 combined with cell-permeable indicators of S383 phosphorylation.

• Super-resolution microscopy: Techniques such as structured illumination microscopy (SIM) or stimulated emission depletion (STED) microscopy to resolve the precise localization at mitotic structures.

• Cell cycle inhibitor studies: Using cell cycle-specific inhibitors to arrest cells at specific phases to examine phosphorylation patterns.

These methodologies help establish the correlation between ELK1 phosphorylation status and its subcellular localization throughout mitosis, revealing functions beyond transcriptional regulation.

What is the relationship between Aurora kinases and Phospho-ELK1 (S383), and how can this interaction be studied?

The relationship between Aurora kinases (particularly Aurora-A) and Phospho-ELK1 (S383) represents an important intersection between cell cycle regulation and transcription factor activity. Research has demonstrated that ELK1 interacts with Aurora-A kinase, and when Aurora inhibitors are used, Phospho-S383-ELK1 fails to localize to spindle poles and remains associated with DNA during mitosis.

To effectively study this interaction:

• Co-immunoprecipitation assays: Using either anti-Aurora-A or anti-Phospho-ELK1 (S383) antibodies to pull down protein complexes and detect the interacting partner by Western blotting.

• Proximity ligation assays (PLA): To visualize and quantify the interaction between Aurora-A and Phospho-ELK1 within intact cells with high specificity and sensitivity.

• Aurora kinase inhibitor studies: Employing selective Aurora-A inhibitors (e.g., MLN8237/Alisertib) to assess changes in Phospho-ELK1 (S383) localization and phosphorylation status.

• In vitro kinase assays: To determine if Aurora-A directly phosphorylates ELK1 at S383 or other residues.

• Knockdown/knockout approaches: Using siRNA/shRNA against Aurora-A or CRISPR-Cas9 gene editing to assess the effect of Aurora-A depletion on ELK1 phosphorylation and localization.

• Cell synchronization combined with inhibitor treatment: To examine phase-specific effects of Aurora inhibition on ELK1 phosphorylation.

Understanding this relationship is crucial for elucidating the mechanistic link between cell cycle progression and ELK1 function, potentially revealing novel therapeutic targets in diseases characterized by dysregulated cell division.

How can Phospho-ELK1 (S383) antibodies be used to investigate cross-talk between MAPK and other signaling pathways?

Phospho-ELK1 (S383) antibodies serve as valuable tools for investigating signaling cross-talk, as ELK1 phosphorylation at S383 represents a convergence point for multiple kinase pathways. Methodological approaches to study this cross-talk include:

• Pathway inhibitor combinations: Sequential or simultaneous application of specific inhibitors targeting MAPK (e.g., U0126 for MEK/ERK), PI3K/AKT (e.g., LY294002), JNK (e.g., SP600125), or p38 (e.g., SB203580) pathways, followed by quantitative assessment of S383 phosphorylation.

• Dual-phosphorylation analysis: Simultaneous detection of Phospho-ELK1 (S383) alongside phosphorylated components of other pathways (e.g., phospho-AKT, phospho-JNK) in the same samples to establish temporal relationships.

• Phosphorylation kinetics: Time-course experiments following stimulation with growth factors or stressors that activate multiple pathways, with quantitative Western blot or ELISA measurement of Phospho-ELK1 (S383) levels.

• Genetic manipulation approaches: Overexpression of constitutively active or dominant-negative components of various signaling pathways to assess their impact on ELK1 S383 phosphorylation.

• Phospho-proteomics: Mass spectrometry-based approaches to simultaneously monitor multiple phosphorylation events on ELK1 and correlate them with specific pathway activations.

This multifaceted approach enables researchers to construct detailed signaling networks and identify novel regulatory connections between canonical pathways that converge on ELK1 as a downstream effector.

What are the common causes of false negative results when detecting Phospho-ELK1 (S383), and how can they be addressed?

Several factors can contribute to false negative results when detecting Phospho-ELK1 (S383):

For each potential issue, control experiments should be conducted in parallel. For instance, detecting another MAPK-dependent phosphorylation event (e.g., phospho-CREB) can confirm pathway activation when Phospho-ELK1 (S383) signal is absent, helping distinguish between experimental and biological causes of negative results.

How should researchers differentiate between specific and non-specific signals when using Phospho-ELK1 (S383) antibodies?

Differentiating specific from non-specific signals is crucial for accurate interpretation of Phospho-ELK1 (S383) antibody results. Robust validation approaches include:

• Phospho-peptide competition: Pre-incubation of the antibody with the phosphorylated peptide immunogen should abolish specific signals while non-specific binding remains.

• Dephosphorylation controls: Treatment of duplicate samples with lambda phosphatase prior to analysis should eliminate specific phospho-dependent signals.

• Stimulation-dependent induction: Specific Phospho-ELK1 (S383) signals should increase following treatments known to activate MAPK pathways (e.g., serum stimulation, growth factors) and decrease with pathway inhibitors.

• Molecular weight verification: On Western blots, specific signal should appear at the predicted molecular weight of 45-46 kDa; bands at substantially different sizes likely represent non-specific interactions.

• Genetic knockdown: siRNA or CRISPR-mediated reduction of ELK1 expression should proportionally decrease the specific phospho-signal.

• Correlation with pathway activation: Specific Phospho-ELK1 (S383) signals should correlate temporally with upstream MAPK activation markers such as phospho-ERK1/2.

• Phospho-site mutant controls: Cells expressing S383A mutant ELK1 should not generate a signal with the phospho-specific antibody.

Implementation of these validation strategies enables confident distinction between specific and artifactual signals across experimental applications.

What are the key experimental design considerations for studying Phospho-ELK1 (S383) in brain tumors and neurological samples?

When investigating Phospho-ELK1 (S383) in brain tumors and neurological samples, researchers should address several special considerations:

• Tissue heterogeneity: Brain samples contain multiple cell types with potentially different ELK1 expression and phosphorylation patterns. Single-cell approaches or microdissection techniques should be considered to resolve cell type-specific signals.

• Post-mortem changes: For human samples, post-mortem interval significantly affects phosphorylation preservation. Phospho-ELK1 (S383) levels should be correlated with post-mortem interval and sample quality metrics.

• Region-specific expression: ELK1 expression and phosphorylation varies across brain regions. Precise anatomical documentation and region-matched controls are essential for meaningful comparisons.

• Pathological correlation: For tumor samples, correlating Phospho-ELK1 (S383) levels with histopathological features, genetic alterations (e.g., MAPK pathway mutations), and clinical outcomes provides contextual relevance.

• Blood-brain barrier considerations: When assessing drug effects on ELK1 phosphorylation, researchers must account for BBB penetration of compounds targeting upstream kinases.

• Fixation protocol optimization: Brain tissues often require modified fixation protocols to balance antigen preservation with tissue architecture maintenance.

• Reference standards: Including positive controls with known phosphorylation status (e.g., mouse brain lysates) alongside experimental samples enhances interpretation reliability.

• Cellular localization assessment: Given ELK1's dual nuclear and mitotic localization, comprehensive imaging throughout tissue sections is necessary to capture the full spectrum of phospho-ELK1 distribution patterns.

These considerations enable robust analysis of Phospho-ELK1 (S383) in neurological contexts, particularly important given the evidence of ELK1 upregulation and phosphorylation in brain tumor cells.

How can Phospho-ELK1 (S383) antibodies be utilized in multi-parameter flow cytometry for cell cycle analysis?

Integrating Phospho-ELK1 (S383) detection into multi-parameter flow cytometry creates powerful opportunities for studying its regulation during cell cycle progression. Methodological approaches include:

• Sample preparation protocol:

  • Fix cells with 4% paraformaldehyde (10 minutes at room temperature)

  • Permeabilize with 90% ice-cold methanol (30 minutes on ice)

  • Block with 0.5% BSA in PBS

  • Incubate with Phospho-ELK1 (S383) antibody (1:200 dilution, 1 hour)

  • Co-stain with cell cycle markers

• Multi-parameter panel design:

  • DNA content (propidium iodide or DAPI)

  • Mitotic marker (phospho-Histone H3)

  • Phospho-ELK1 (S383)

  • Optional: Additional cell cycle regulatory proteins (Cyclin B1, Aurora kinases)

• Gating strategy:

  • Initial gating on FSC/SSC to identify intact cells

  • Single cell selection using pulse-width discrimination

  • Cell cycle phase identification based on DNA content

  • Analysis of Phospho-ELK1 (S383) intensity within each cell cycle phase

• Controls for flow cytometry optimization:

  • Unstimulated vs. stimulated samples (serum or growth factors)

  • Phosphatase-treated negative controls

  • MAPK pathway inhibitor (U0126) treated samples

  • Isotype controls for antibody specificity

This approach enables quantitative assessment of ELK1 phosphorylation dynamics throughout the cell cycle at the single-cell level, revealing heterogeneity within populations and correlations with other signaling events.

What are the emerging techniques for studying the spatial and temporal dynamics of Phospho-ELK1 (S383) in live cells?

Cutting-edge approaches for monitoring Phospho-ELK1 (S383) dynamics in living cells include:

• Phospho-specific FRET biosensors:

  • Engineered ELK1 constructs containing appropriate fluorophore pairs

  • FRET signal changes upon S383 phosphorylation

  • Enables real-time visualization of phosphorylation events

  • Provides subcellular resolution of kinase activity

• Optogenetic control of MAPK pathway components:

  • Light-activated RAF or MEK variants to trigger pathway activation

  • Paired with fluorescent Phospho-ELK1 (S383) reporters

  • Allows precise temporal control of phosphorylation events

  • Facilitates study of activation/deactivation kinetics

• CRISPR-based ELK1 endogenous tagging:

  • Knock-in of fluorescent tags at the endogenous ELK1 locus

  • Combined with phospho-specific antibody fragments

  • Maintains physiological expression levels

  • Avoids artifacts of overexpression systems

• Advanced imaging platforms:

  • Lattice light-sheet microscopy for 3D visualization with minimal phototoxicity

  • High-content imaging systems for population-level quantification

  • Correlative light and electron microscopy for ultrastructural context

• Bimolecular fluorescence complementation:

  • Split fluorescent protein fragments attached to ELK1 and phospho-binding domains

  • Fluorescence reconstitution upon S383 phosphorylation

  • Enables visualization of phosphorylation-dependent interactions

These emerging techniques provide unprecedented insights into the spatiotemporal regulation of ELK1 phosphorylation and its relationship to subcellular localization throughout the cell cycle, particularly during mitotic phases where traditional immunofluorescence approaches may have limitations.