Phospho-VASP (Ser157) Antibody

What is VASP and what significance does phosphorylation at Ser157 have?

VASP (Vasodilator-stimulated phosphoprotein) is a cellular protein highly expressed in platelets and involved in cell motility, migration, and adhesion processes . Phosphorylation of VASP at serine 157 (Ser157) is a critical post-translational modification primarily mediated by protein kinase A (PKA) and serves as a key regulatory mechanism in various cellular pathways. This phosphorylation event is particularly important in:

• Cardiovascular research, where VASP phosphorylation status affects platelet activation

• Oncology studies, where cell migration pathways involve VASP regulation

• Inflammation research, where VASP participates in immune cell functions

The phosphorylation state at Ser157 specifically serves as a direct indicator of PKA activation in cells, making it a valuable biomarker for monitoring signaling pathway activity .

What are the common applications for Phospho-VASP (Ser157) antibodies?

Phospho-VASP (Ser157) antibodies are versatile tools in research laboratories with several established applications:

These applications collectively enable researchers to study VASP phosphorylation in diverse experimental systems, from purified proteins to intact tissues .

How should I validate the specificity of a Phospho-VASP (Ser157) antibody?

Validation of phospho-specific antibodies requires a multi-faceted approach to ensure experimental reliability:

• Positive and negative controls: Use samples with known phosphorylation status, including:

  • Cells treated with PKA activators (positive control)

  • Untreated cells (negative or baseline control)

  • Cells treated with phosphatase inhibitors to preserve phosphorylation

• Molecular weight verification: Confirm band detection at the expected molecular weight (approximately 50 kDa for phosphorylated VASP)

• Comparing multiple detection methods: Cross-validate results using:

  • Different antibody clones targeting the same phospho-site

  • Alternative detection methods like HTRF assays

  • Mass spectrometry for definitive phosphorylation site confirmation

• Phosphatase treatment: Treat a portion of your positive control sample with lambda phosphatase before analysis to demonstrate phospho-specificity.

This comprehensive validation ensures confidence in experimental results and prevents misinterpretation of antibody signals .

What species reactivity should I consider when selecting a Phospho-VASP (Ser157) antibody?

Species reactivity is a crucial consideration for experimental planning. Available antibodies show distinct cross-reactivity profiles:

• Confirmed reactivity: Most commercial Phospho-VASP (Ser157) antibodies reliably detect human, mouse, and rat proteins

• Extended reactivity: Some antibodies demonstrate additional reactivity with monkey (Mk) and guinea pig (GP) samples

• Predicted reactivity: Computational analysis suggests potential cross-reactivity with pig, bovine, sheep, and dog samples, though experimental validation is recommended

When working with less common research species, it's advisable to perform preliminary validation tests, as the sequence conservation around the Ser157 site influences antibody recognition . The choice of antibody should align with the research model to ensure reliable detection and meaningful results.

What are the optimal sample preparation conditions for Phospho-VASP (Ser157) detection?

Proper sample preparation is essential for maintaining phosphorylation status and achieving reliable detection:

• Lysis conditions:

  • Use phosphatase inhibitors (sodium fluoride, sodium orthovanadate, β-glycerophosphate) in all buffers

  • Maintain cold temperatures (4°C) throughout processing

  • Use a buffer containing 1% Triton X-100, 150 mM NaCl, and 50 mM Tris pH 7.4 for effective protein extraction

• Protein quantification:

  • Normalize all samples to equal protein concentration

  • Typically load 10-20 μg of total protein for Western blot applications

• For HTRF assays:

  • Sample volume of 16 μL is recommended

  • Follow two-plate protocol for optimal results: culture cells in 96-well format, then transfer lysates to 384-well detection plates

• Storage considerations:

  • Analyze samples immediately when possible

  • For short-term storage, keep at -20°C

  • For long-term preservation, store at -80°C with additional phosphatase inhibitors

Careful attention to these parameters helps preserve the phosphorylation state and increases detection reliability .

How can I effectively troubleshoot inconsistent phospho-VASP (Ser157) signals in Western blot analysis?

Inconsistent phospho-VASP (Ser157) signals can arise from several factors that require systematic troubleshooting:

• Phosphorylation preservation issues:

  • Ensure complete phosphatase inhibition in all buffers

  • Minimize time between cell harvesting and protein denaturation

  • Consider using phosphatase inhibitor cocktails at higher concentrations

• Antibody-specific considerations:

  • Test different antibody dilutions (1:500 to 1:2000) to optimize signal-to-noise ratio

  • Extend primary antibody incubation time (overnight at 4°C)

  • Try different blocking agents (BSA vs. non-fat milk) as milk contains phosphatases

• Gel electrophoresis parameters:

  • Use freshly prepared samples when possible

  • Ensure complete protein denaturation (95°C for 5 minutes in Laemmli buffer)

  • Consider using Phos-tag™ acrylamide gels for enhanced separation of phosphorylated species

• Detection system optimization:

  • For chemiluminescence, use freshly prepared ECL substrate

  • For fluorescence-based detection, minimize exposure to light

  • Consider using signal enhancement systems for low-abundance targets

• Experimental controls:

  • Always run positive controls (PKA-activated samples)

  • Include total VASP antibody detection on stripped or parallel blots to normalize for total protein levels

For particularly challenging samples, consider alternative approaches like the HTRF assay, which offers a no-wash format that may preserve phosphorylation status more effectively .

What are the advantages and limitations of HTRF assays versus Western blot for phospho-VASP (Ser157) detection?

Choosing between HTRF and Western blot methodologies involves weighing several factors:

HTRF Assays Advantages:

• Higher throughput: Enables analysis of 96 or 384 samples simultaneously

• Quantitative precision: Provides direct quantification without densitometry

• Simplified workflow: Plate-based format eliminates gel electrophoresis and transfer steps

• No-wash format: Reduces sample manipulation, potentially preserving phosphorylation status

• Speed: Results typically available within 4 hours from sample preparation

HTRF Assays Limitations:

• Higher cost per sample: Specialized reagents and detection equipment required

• Limited to known targets: Cannot discover novel phosphorylation sites

• Sample compatibility: May have matrix effects in complex samples

• Fixed specificity: Dependent on the predefined antibody pair specificity

Western Blot Advantages:

• Visual confirmation: Provides molecular weight information to confirm specificity

• Flexibility: Can detect multiple phosphorylation sites with stripping and reprobing

• Widespread accessibility: Standard equipment available in most research labs

• Lower cost per sample: More economical for small sample numbers

• Established technique: Extensive literature references for comparison

Western Blot Limitations:

• Labor intensive: Multiple manual steps increase variability

• Semi-quantitative: Densitometry has limitations in linear range

• Lower throughput: Typically processing 10-20 samples per gel

• Transfer efficiency variables: Protein loss during transfer affects quantification

• Longer time to results: Typically takes 1-2 days to complete

How can I design experiments to distinguish between PKA- and PKG-mediated phosphorylation of VASP at Ser157?

Distinguishing between PKA- and PKG-mediated phosphorylation requires careful experimental design:

• Selective pathway activation:

  • For PKA: Use forskolin (adenylyl cyclase activator) or cAMP analogs (e.g., 8-Br-cAMP)

  • For PKG: Use NO donors (e.g., SNAP, SNP) or cGMP analogs (e.g., 8-Br-cGMP)

• Pathway-specific inhibitors:

  • PKA inhibition: H-89, PKI (Protein Kinase A Inhibitor)

  • PKG inhibition: KT5823, Rp-8-Br-PET-cGMPS

  • Use inhibitors in combination with activators to confirm pathway specificity

• Genetic approaches:

  • siRNA knockdown of PKA or PKG catalytic subunits

  • CRISPR/Cas9 knockout of pathway components

  • Overexpression of dominant-negative kinase mutants

• Time-course analysis:

  • PKA typically shows more rapid phosphorylation kinetics

  • Monitor phosphorylation at early timepoints (1-5 minutes) to distinguish initial kinase activity

• Parallel site analysis:

  • While both PKA and PKG can phosphorylate VASP at Ser157, PKG preferentially phosphorylates Ser239

  • Monitor Ser157 and Ser239 phosphorylation simultaneously to infer kinase activity

• In vitro kinase assays:

  • Perform in vitro kinase reactions with purified PKA and PKG

  • Use recombinant VASP as substrate

  • Compare phosphorylation efficiency at different ATP concentrations

By combining these approaches, researchers can build a comprehensive profile of kinase-specific VASP phosphorylation patterns and confidently attribute observed effects to specific signaling pathways .

What experimental controls are crucial when studying drug-induced changes in VASP phosphorylation?

Robust experimental design for drug studies requires comprehensive controls:

• Baseline controls:

  • Vehicle-only treatment matching the drug solvent (e.g., DMSO, ethanol)

  • Time-matched untreated samples to account for temporal variations

  • Concentration-matched inactive analogs of test compounds

• Positive controls:

  • Direct PKA activators (8-Br-cAMP, forskolin) for maximum Ser157 phosphorylation

  • Known pathway activators with well-characterized effects on VASP phosphorylation

• Negative controls:

  • Pathway inhibitor pre-treatment to block phosphorylation

  • Phosphatase treatment of cell lysates to remove all phosphorylation

  • Cells with VASP knockdown/knockout to confirm antibody specificity

• Dose-response controls:

  • Multiple drug concentrations to establish concentration-dependency

  • Time-course analysis to capture kinetics of phosphorylation changes

• Pathway validation controls:

  • Parallel measurement of upstream components (e.g., cAMP levels, PKA activity)

  • Monitoring additional PKA substrates to confirm pathway activation

  • Use of pathway-specific inhibitors to verify mechanism

• Cell state controls:

  • Proliferating vs. serum-starved conditions

  • Confluent vs. sub-confluent cultures

  • Different cell cycle phases if relevant

• Total protein controls:

  • Parallel detection of total VASP protein levels

  • Normalization to loading controls (β-actin, GAPDH)

  • Evaluation of sample-to-sample variation in total protein content

Including these controls enables confident interpretation of drug effects on VASP phosphorylation pathways and facilitates comparison between different experimental conditions and compounds .

How can I effectively study VASP phosphorylation dynamics in primary cells versus cell lines?

Studying VASP phosphorylation in different cellular systems requires tailored approaches:

Primary Cell Considerations:

• Isolation and culture optimization:

  • Minimize stress during isolation that may activate stress kinases

  • Allow recovery period (4-24 hours) after isolation before stimulation

  • Use physiologically relevant culture conditions (e.g., specific ECM components)

• Donor variation management:

  • Use multiple donors to account for genetic variability

  • Consider pooling samples to establish baseline responses

  • Document donor characteristics that may influence signaling (age, sex, disease state)

• Signal detection optimization:

  • Higher antibody concentrations may be needed (1:500 dilution for Western blot)

  • Increase cell numbers to compensate for potentially lower VASP expression

  • Consider more sensitive detection methods like HTRF

• Experimental timing:

  • Primary cells typically have limited passages before phenotypic drift

  • Ensure consistent passage number or days in culture

  • Perform experiments within the window of stable phenotype

Cell Line Considerations:

• Line selection guidance:

  • Choose lines with confirmed VASP expression

  • Consider tissue origin relevance to research question

  • Verify PKA/PKG pathway functionality

• Protocol optimization:

  • Standard 1:1000 antibody dilutions often sufficient

  • Shorter stimulation times may be effective due to homogeneous responses

  • Consider creating stable reporter cell lines for repeated studies

• Cell density effects:

  • VASP phosphorylation can vary with confluency

  • Standardize seeding density and time before experiments

  • Document growth phase (log, plateau) during experiments

Comparative Analysis Strategies:

• Direct comparison approaches:

  • Process primary cells and cell lines in parallel with identical protocols

  • Normalize to total VASP expression when comparing across cell types

  • Consider creating ratio measurements (phospho/total) to account for expression differences

• Protocol adaptations for comparative studies:

  • Cell type-specific lysis buffers may be required

  • Adjust stimulation times based on response kinetics in each system

  • Consider using the same detection platform (e.g., HTRF) for both systems to minimize method-based variation

This comprehensive approach enables meaningful comparison of VASP phosphorylation dynamics between research models while accounting for inherent biological differences .

How can I optimize Western blot conditions specifically for Phospho-VASP (Ser157) detection?

Western blot optimization for phospho-VASP requires attention to several technical parameters:

• Sample preparation refinements:

  • Use RIPA buffer supplemented with 50 mM NaF, 2 mM Na₃VO₄, and 10 mM β-glycerophosphate

  • Sonicate lysates briefly (3 × 5 seconds) to shear DNA and reduce viscosity

  • Heat samples at 70°C for 10 minutes rather than 95°C to minimize phosphate hydrolysis

• Gel selection and running conditions:

  • Use 10-12% polyacrylamide gels for optimal resolution

  • Consider gradient gels (4-15%) to capture both phosphorylated forms (46-50 kDa)

  • Maintain gel running temperature below 25°C to preserve phosphorylation

• Transfer optimization:

  • Use PVDF membranes (0.45 μm pore size) for higher protein binding capacity

  • Include methanol (10-20%) in transfer buffer to remove SDS

  • Opt for wet transfer systems for larger proteins like VASP

• Blocking considerations:

  • Use 5% BSA in TBS-T rather than milk (milk contains phosphatases)

  • Block for 1 hour at room temperature or overnight at 4°C

  • Consider commercial phospho-protein specific blocking buffers

• Antibody conditions:

  • Optimal dilution typically 1:1000 in 5% BSA/TBS-T

  • Incubate overnight at 4°C with gentle rocking

  • Wash extensively (5 × 5 minutes) with TBS-T before secondary antibody

• Detection enhancements:

  • Use high-sensitivity ECL substrates for enhanced detection

  • Consider fluorescent secondary antibodies for better quantitative range

  • Optimize exposure times to prevent saturation for accurate quantification

• Controls and normalization:

  • Run phosphatase-treated sample as negative control

  • Detect total VASP on the same membrane after stripping

  • Include loading control (β-actin, GAPDH) for normalization

These optimized conditions help maximize signal specificity and sensitivity while preserving the phosphorylation status of VASP .

What are the key considerations for multiplexing phospho-VASP detection with other signaling proteins?

Multiplexing phospho-VASP detection requires strategic planning to avoid interference and maximize data quality:

• Antibody compatibility assessment:

  • Select antibodies from different host species (e.g., rabbit anti-phospho-VASP with mouse anti-other targets)

  • Verify no cross-reactivity between antibodies

  • Consider antibodies directly conjugated to different fluorophores to eliminate secondary antibody issues

• Protein size considerations:

  • For targets with similar molecular weights, use different colored detection systems

  • For Western blots, cut membranes horizontally between size ranges when necessary

  • Consider sequential probing after complete stripping for overlapping targets

• Phosphorylation preservation strategies:

  • Maintain consistent phosphatase inhibitor cocktails throughout

  • Minimize freeze-thaw cycles of samples

  • Process all multiplexed samples simultaneously under identical conditions

• Detection system selection:

  • For fluorescent detection: Use spectrally distinct fluorophores with minimal overlap

  • For chemiluminescence: Use sequential detection with substrate removal between steps

  • For HTRF assays: Verify absence of energy transfer between unrelated antibody pairs

• Data analysis adaptations:

  • Establish individual baseline controls for each phospho-protein

  • Apply appropriate background subtraction for each target

  • Consider ratiometric analysis (phospho/total) for each protein separately

• Validation approaches:

  • Confirm multiplexed results with single-plex detection

  • Verify consistency across technical replicates

  • Include pathway-specific positive controls for each target

• Practical implementation in different platforms:

  • Western blot: Simultaneous transfer followed by sequential probing

  • Immunofluorescence: Multilabel protocols with appropriate blocking between steps

  • HTRF: Separate wells or custom assay development for orthogonal targets

Effective multiplexing enables efficient use of limited samples and provides valuable insight into signaling network relationships .

How does the HTRF detection method for phospho-VASP (Ser157) work, and what are its technical requirements?

The HTRF (Homogeneous Time-Resolved Fluorescence) detection system represents an advanced approach for phospho-VASP (Ser157) quantification:

Principle of Operation:

• Dual antibody recognition system:

  • First antibody: Specifically binds to the phosphorylated Ser157 motif on VASP

  • Second antibody: Recognizes VASP protein regardless of phosphorylation state

• FRET-based detection mechanism:

  • First antibody is labeled with a donor fluorophore

  • Second antibody carries an acceptor fluorophore

  • When both antibodies bind to phospho-VASP, the proximity enables energy transfer (FRET)

  • Signal intensity directly correlates with phospho-VASP concentration

• Time-resolved measurement:

  • Long-lived fluorescence of lanthanide donor eliminates background fluorescence

  • Signal measured after a time delay to enhance signal-to-noise ratio

  • Enables detection in complex biological matrices

Technical Requirements:

• Instrumentation needs:

  • Microplate reader with time-resolved fluorescence capability

  • Excitation filter: 320-340 nm

  • Emission filters: 615 nm (donor) and 665 nm (acceptor)

  • Temperature control (room temperature detection)

• Sample considerations:

  • Minimum volume: 16 μL per well

  • Cell number: Typically 10,000-100,000 cells per well

  • Compatible with adherent and suspension cells

  • Works with diverse biological samples (cell lines, primary cells)

• Assay configuration options:

  • Two-plate protocol: Culture cells in 96-well plate, transfer lysates to 384-well detection plate

  • Single-plate protocol: Culture, stimulate and detect in the same plate

  • Both approaches eliminate the need for gels, electrophoresis, or transfer steps

• Technical advantages:

  • No-wash format minimizes sample manipulation

  • Homogeneous assay reduces variability

  • Plate-based format enables higher throughput

  • Directly quantitative without densitometry

  • Results available within hours rather than days

The HTRF method represents a significant advance for researchers requiring quantitative phospho-VASP analysis, particularly in screening applications or when processing numerous samples simultaneously .

What are the best methods for quantifying and statistically analyzing phospho-VASP (Ser157) data?

Robust quantification and statistical analysis of phospho-VASP data requires methodical approaches:

Quantification Methods:

• Western blot densitometry:

  • Use linear range calibration curves with recombinant standards

  • Apply background subtraction consistently across all samples

  • Normalize phospho-VASP signal to total VASP signal

  • Report as fold change relative to control conditions

  • Use analysis software that prevents saturation during image acquisition

• HTRF signal analysis:

  • Calculate ratio of acceptor (665 nm) to donor (615 nm) emission

  • Use delta F% calculation: [(Sample ratio - Negative control ratio) / Negative control ratio] × 100

  • Create standard curves with known concentrations of phospho-peptides

  • Express results as absolute concentration when standards are available

• Immunofluorescence quantification:

  • Measure mean pixel intensity within defined cellular regions

  • Use cell-by-cell analysis rather than field averages

  • Normalize to total VASP staining in dual-labeled samples

  • Consider subcellular distribution patterns beyond total intensity

Statistical Analysis Approaches:

• Experimental design considerations:

  • Determine appropriate sample size through power analysis

  • Include biological replicates (different cell preparations)

  • Perform technical replicates (multiple measurements of same sample)

  • Design experiments with proper controls for normalization

• Parametric vs. non-parametric testing:

  • Test data for normality using Shapiro-Wilk or Kolmogorov-Smirnov tests

  • For normally distributed data: t-tests (two groups) or ANOVA (multiple groups)

  • For non-normally distributed data: Mann-Whitney U (two groups) or Kruskal-Wallis (multiple groups)

• Multiple comparison corrections:

  • Apply Bonferroni correction for conservative approach

  • Consider False Discovery Rate (FDR) methods for multiple comparisons

  • Use Tukey or Dunnett post-hoc tests following ANOVA

• Advanced analytical approaches:

  • For time-course studies: repeated measures ANOVA or mixed-effects models

  • For dose-response data: non-linear regression to determine EC50 values

  • For complex experimental designs: multifactorial ANOVA or linear mixed models

• Visualization best practices:

  • Present individual data points alongside means/medians

  • Include error bars representing standard deviation or standard error

  • Use consistent scales when comparing different experimental conditions

  • Consider log transformation for data spanning multiple orders of magnitude

This comprehensive approach to quantification and statistical analysis ensures reliable interpretation of phospho-VASP data across different experimental platforms .

How can phospho-VASP (Ser157) be used as a biomarker in cardiovascular research?

Phospho-VASP (Ser157) serves as a valuable biomarker in cardiovascular research with multiple applications:

• Platelet function assessment:

  • VASP phosphorylation status directly correlates with platelet reactivity

  • Increased phospho-VASP (Ser157) indicates suppressed platelet activation

  • Monitoring phospho-VASP levels helps evaluate antiplatelet therapy efficacy

  • Allows personalized dosing of P2Y₁₂ inhibitors (e.g., clopidogrel)

• Endothelial function studies:

  • Phospho-VASP levels reflect NO/cGMP pathway integrity in endothelial cells

  • Decreased phosphorylation may indicate endothelial dysfunction

  • Changes in phosphorylation kinetics can reveal impaired signaling mechanisms

  • Useful for evaluating vascular health in disease models

• Vascular smooth muscle research:

  • VASP phosphorylation influences actin cytoskeleton organization

  • Changes in phospho-VASP correlate with alterations in vascular tone

  • Decreased phosphorylation associates with vascular remodeling in hypertension

  • Monitors beneficial effects of vasoactive therapies

• Ischemia-reperfusion studies:

  • Phospho-VASP levels change dynamically during cardiac ischemia-reperfusion

  • Protective interventions often maintain or restore VASP phosphorylation

  • Serves as a surrogate marker for cardioprotective signaling pathway integrity

  • Helps evaluate potential cardioprotective agents

• Atherosclerosis research applications:

  • Altered VASP phosphorylation occurs in atherosclerotic lesions

  • Changes in phospho-VASP patterns during plaque progression

  • May serve as a marker for plaque stability assessment

  • Potential target for anti-atherosclerotic interventions

• Methodological approaches:

  • Western blot analysis of tissue or cell lysates for mechanistic studies

  • Flow cytometry for platelet function analysis in clinical samples

  • HTRF assays for high-throughput drug screening applications

  • Immunohistochemistry for spatial distribution in vascular tissues

These applications collectively establish phospho-VASP (Ser157) as an informative biomarker spanning from basic cardiovascular research to potential clinical applications .

What role does phospho-VASP (Ser157) play in cancer research and potential therapeutic applications?

Phospho-VASP (Ser157) has emerging significance in cancer biology and therapeutic development:

• Cell migration and metastasis mechanisms:

  • VASP phosphorylation regulates actin cytoskeleton dynamics

  • Changes in phospho-VASP status influence cancer cell motility

  • Altered phosphorylation patterns correlate with metastatic potential

  • Phospho-VASP serves as an indicator of migratory phenotype acquisition

• Tumor cell invasion pathways:

  • Phospho-VASP (Ser157) affects formation of membrane protrusions

  • Modulates extracellular matrix degradation capacity

  • Influences invadopodia formation in invasive cancer cells

  • Serves as a downstream effector of pro-invasive signaling cascades

• Cancer type-specific patterns:

  • Breast cancer: Increased phospho-VASP in aggressive subtypes

  • Colorectal cancer: Phosphorylation changes correlate with progression

  • Lung cancer: Altered VASP phosphorylation in metastatic disease

  • Leukemia: Dynamic phosphorylation during differentiation therapy

• Signal transduction pathway integration:

  • PKA-mediated VASP phosphorylation connects cAMP signaling to cytoskeletal regulation

  • Serves as a convergence point for multiple oncogenic signals

  • Changes in phospho-VASP indicate alterations in upstream kinase activities

  • Provides insights into cancer-specific signaling adaptations

• Therapeutic targeting approaches:

  • Direct targeting: Compounds affecting VASP phosphorylation

  • Indirect approaches: Modulating upstream PKA activity

  • Combination strategies: Enhancing cytoskeletal-targeting drug efficacy

  • Biomarker application: Monitoring therapy-induced signaling changes

• Research methodology considerations:

  • Immunohistochemical analysis of tissue microarrays for clinical correlations

  • Live-cell imaging with phospho-specific antibodies for dynamic studies

  • HTRF assays for high-throughput drug screening campaigns

  • Western blot analysis for basic mechanistic investigations

These diverse applications highlight the potential of phospho-VASP (Ser157) as both a mechanistic research tool and a potential therapeutic target in cancer research .

How can I design experiments to investigate the relationship between VASP phosphorylation at different sites (Ser157, Ser239) and cellular function?

Investigating the functional consequences of site-specific VASP phosphorylation requires sophisticated experimental design:

• Site-specific phosphorylation induction:

  • Ser157 preferential activation: Use PKA-specific activators (forskolin, 8-Br-cAMP)

  • Ser239 preferential activation: Use PKG-specific activators (NO donors, 8-Br-cGMP)

  • Dual site regulation: Manipulate both pathways sequentially or simultaneously

  • Time-course studies to capture site-specific kinetics

• Molecular tools for site-specific analysis:

  • Phospho-site mutants: S157A, S239A (non-phosphorylatable) and S157D, S239D (phosphomimetic)

  • Expression vectors for wild-type and mutant rescue experiments

  • CRISPR/Cas9 knock-in of site-specific mutations

  • Site-specific phospho-antibodies for parallel detection

• Functional phenotype assays:

  • Cell migration: Wound healing, transwell, and single-cell tracking assays

  • Cytoskeletal dynamics: Live F-actin imaging, filopodia/lamellipodia quantification

  • Adhesion strength: Measuring cell-substrate adhesion forces

  • Protein interactions: Co-immunoprecipitation with actin-binding partners

• Dual-site phosphorylation assessment:

  • Sequential immunoblotting with site-specific antibodies

  • Phospho-protein enrichment followed by mass spectrometry

  • Phos-tag gel electrophoresis to separate differentially phosphorylated species

  • Dual-color immunofluorescence with site-specific antibodies

• Quantitative analysis approaches:

  • Phosphorylation stoichiometry measurements

  • Correlation analysis between phosphorylation level and functional outcomes

  • Mathematical modeling of site-specific phosphorylation dynamics

  • Spatial distribution analysis of differentially phosphorylated VASP pools

• Experimental matrix design:

  Condition	Expected Ser157	Expected Ser239	Predicted Phenotype
  Basal	Low	Low	Baseline cytoskeletal dynamics
  PKA activation	High	Low	Enhanced cell migration
  PKG activation	Low	High	Reduced cell migration
  Dual activation	High	High	Complex phenotype requiring analysis
  S157A mutant	None	Variable	Impaired PKA-dependent functions
  S239A mutant	Variable	None	Impaired PKG-dependent functions
  Dual S157A/S239A	None	None	Maximal functional disruption

This comprehensive experimental approach enables researchers to dissect the specific contributions of each phosphorylation site to VASP-mediated cellular functions, revealing site-specific regulatory mechanisms .

What are the latest advances in understanding the relationship between VASP phosphorylation and cellular mechanotransduction?

Emerging research has revealed complex relationships between VASP phosphorylation and mechanotransduction processes:

• Mechanical force sensing:

  • Phospho-VASP localization changes under different mechanical stimuli

  • Ser157 phosphorylation responds dynamically to substrate stiffness changes

  • Mechanical stretching induces rapid VASP phosphorylation patterns

  • Flow-induced shear stress triggers site-specific phosphorylation responses

• Focal adhesion dynamics:

  • Phospho-VASP (Ser157) enrichment at mechanosensitive focal adhesions

  • Phosphorylation status influences focal adhesion maturation and turnover

  • Force-dependent recruitment of differentially phosphorylated VASP species

  • Altered phosphorylation kinetics in response to mechanical perturbations

• Cytoskeletal tension integration:

  • VASP phosphorylation modulates actin filament elongation rates under tension

  • Ser157 phosphorylation affects force transmission through actin networks

  • Mechanical feedback loops involving VASP phosphorylation and cytoskeletal tension

  • Altered mechanical properties of cells with phospho-site mutations

• Methodological innovations:

  • Force microscopy combined with phospho-specific immunolabeling

  • Tension sensors integrated with VASP constructs for real-time analysis

  • Micropattern substrates to control cell geometry and force distribution

  • Advanced imaging techniques for nanoscale phosphorylation distribution

• Physiological implications:

  • Vascular adaptation to flow patterns involves VASP phosphorylation dynamics

  • Cardiac muscle response to mechanical load includes VASP phosphorylation changes

  • Cellular adaptations to matrix stiffness mediated by VASP phosphorylation status

  • Mechanoprotection mechanisms involving phospho-VASP regulation

These emergent findings position phospho-VASP (Ser157) at the intersection of biochemical signaling and mechanical force sensing, opening new avenues for understanding cellular adaptation to physical environments .

How is phospho-VASP research being applied to novel therapeutic target identification?

Phospho-VASP research is generating promising therapeutic applications across multiple disease contexts:

• Cardiovascular therapeutic targets:

  • Antiplatelet therapies: Monitoring phospho-VASP as efficacy biomarker

  • Endothelial dysfunction: Targeting pathways that restore VASP phosphorylation

  • Vascular remodeling: Modulating VASP phosphorylation to prevent pathological changes

  • Heart failure: Exploiting VASP-mediated cardioprotective mechanisms

• Cancer therapeutic avenues:

  • Migration inhibition: Compounds that modulate VASP phosphorylation state

  • Metastasis prevention: Targeting VASP-dependent invasive processes

  • Combination approaches: Enhancing cytoskeletal-targeting chemotherapies

  • Biomarker application: Stratifying patients for specific treatment approaches

• Inflammatory disease applications:

  • Leukocyte adhesion: Modulating VASP-dependent endothelial barrier function

  • Immune cell migration: Targeting phospho-VASP in specific leukocyte populations

  • Resolution phase enhancement: Promoting VASP phosphorylation states that support tissue repair

  • Vascular leak prevention: Stabilizing endothelial junctions through VASP regulation

• Drug discovery approaches:

  • High-throughput screening: Using HTRF phospho-VASP assays to identify novel compounds

  • Structure-based design: Targeting VASP domains involved in phosphorylation-dependent interactions

  • Pathway modulation: Identifying upstream regulators of VASP phosphorylation

  • Repurposing strategies: Identifying approved drugs that affect VASP phosphorylation

• Translational research applications:

  • Patient stratification: Using phospho-VASP patterns to predict treatment response

  • Therapy monitoring: Sequential phospho-VASP assessment during treatment

  • Combination therapy rationale: Targeting complementary pathways affecting VASP phosphorylation

  • Precision medicine approaches: Matching treatments to phospho-VASP profiles

These diverse therapeutic applications highlight the translational potential of phospho-VASP research, bridging fundamental cellular mechanisms with clinical applications across multiple disease contexts .

What are the most important considerations for researchers planning phospho-VASP (Ser157) experiments?

When planning phospho-VASP (Ser157) experiments, researchers should consider several critical factors to ensure reliable and meaningful results:

By addressing these considerations systematically, researchers can design robust experiments that generate reliable and biologically meaningful data on phospho-VASP (Ser157) regulation and function across diverse research contexts .

What future directions are emerging in phospho-VASP research methodology and applications?

Phospho-VASP research is evolving rapidly with several promising future directions:

• Methodological innovations:

  • Single-cell phospho-proteomics for heterogeneity analysis

  • Live-cell biosensors for real-time phosphorylation monitoring

  • Mass cytometry (CyTOF) for multi-parameter phospho-VASP analysis

  • AI-assisted image analysis for complex phosphorylation pattern recognition

  • Microfluidic platforms for dynamic phosphorylation studies under controlled conditions

• Multi-site phosphorylation analysis:

  • Integrated assessment of all VASP phosphorylation sites simultaneously

  • Computational modeling of phosphorylation site interactions

  • Structural studies of phosphorylation-induced conformational changes

  • Functional consequences of combinatorial phosphorylation patterns

  • Temporal ordering of multi-site phosphorylation events

• Systems biology integration:

  • Network analysis of VASP interactome based on phosphorylation state

  • Pathway modeling incorporating VASP phosphorylation dynamics

  • Multi-omics approaches connecting phospho-VASP to broader cellular responses

  • Mathematical modeling of cytoskeletal dynamics regulated by phospho-VASP

• Translational research trajectories:

  • Development of phospho-VASP-based companion diagnostics

  • Therapeutic targeting of specific VASP phosphorylation states

  • Patient stratification based on phospho-VASP profiles

  • Monitoring treatment efficacy using phospho-VASP as biomarker

• Emerging biological contexts:

  • Neurobiology: Phospho-VASP in neuronal development and plasticity

  • Immunology: VASP phosphorylation in immune cell function

  • Developmental biology: Role in tissue morphogenesis and cell differentiation

  • Aging research: Changes in VASP phosphorylation with cellular senescence

• Advanced technological applications:

  • Organ-on-chip systems incorporating phospho-VASP monitoring

  • CRISPR-based phosphorylation site editing for functional studies

  • Phospho-VASP status in 3D tissue models and organoids

  • High-content screening platforms for phospho-VASP modulators