Phospho-MYLK (Y464) Antibody

What is MYLK and why is Y464 phosphorylation important?

MYLK (Myosin Light Chain Kinase) is a serine/threonine kinase that phosphorylates myosin regulatory light chains to facilitate myosin-actin interaction and contractile activity . The protein exists in multiple isoforms, with the non-muscle MLCK (nmMLCK) being particularly important in endothelial cells . Y464 phosphorylation represents a critical regulatory site for nmMLCK enzymatic activation . This phosphorylation event is essential for proper spatial localization of nmMLCK to the cell periphery, where it can direct increases in myosin light chain (MLC) phosphorylation . The phosphorylation at Y464 is particularly significant because it coincides with increases in trans-endothelial electrical resistance (TER) and elastic modulus, reflecting enhanced vascular barrier integrity .

What are the optimal applications for Phospho-MYLK (Y464) Antibody?

Phospho-MYLK (Y464) Antibody has been validated for several research applications:

For immunohistochemistry applications, sodium citrate pH 6.0 is recommended for antigen retrieval (>98°C, 20min) . The antibody has been specifically tested and validated with human tissue samples, with particular effectiveness in detecting endogenous levels of MYLK when phosphorylated at Tyr464 .

How should researchers store and handle Phospho-MYLK (Y464) Antibody?

For optimal antibody performance, follow these storage guidelines:

• Long-term storage: Store at -20°C for up to one year or at -80°C for extended periods

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles as this can degrade antibody activity

• The antibody is typically supplied in liquid form in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide

When handling the antibody for experiments, it's advisable to aliquot the stock solution to minimize freeze-thaw cycles and maintain consistent antibody performance across experiments.

What species reactivity does Phospho-MYLK (Y464) Antibody demonstrate?

Based on validation studies, Phospho-MYLK (Y464) Antibody reactivity varies by manufacturer:

• Most commercially available antibodies are reactive to human MYLK when phosphorylated at Y464

• Some antibody products show broader reactivity for MYLK protein (non-phospho-specific) across human, mouse, and rat species, as demonstrated by Western blot analysis

• The antibody specifically recognizes endogenous levels of MYLK only when phosphorylated at Tyr464 position

Researchers should verify species cross-reactivity for their specific experimental needs, as the immunogen typically consists of a synthesized phospho-peptide derived from human MYLK around the phosphorylation site of Y464 .

What controls should be included when using Phospho-MYLK (Y464) Antibody?

For rigorous experimental design with phospho-specific antibodies, include the following controls:

• Positive control: Lysates from cells treated with sphingosine 1-phosphate (S1P), which induces Y464 phosphorylation

• Negative control: Samples treated with phosphatase to remove phosphorylation

• Peptide competition assay: Using the immunizing phosphopeptide to confirm specificity

• Non-phosphorylated control: Comparing with total MYLK antibody staining

• Isotype control: Using rabbit IgG at equivalent concentration to assess non-specific binding

When conducting immunohistochemistry, include both positive and negative tissue controls to validate staining patterns and optimize antibody dilution ratios.

How do MYLK coding polymorphisms affect Y464 phosphorylation in inflammatory lung diseases?

MYLK coding single nucleotide polymorphisms (SNPs) have significant functional impacts on Y464 phosphorylation with implications for lung inflammatory diseases:

• Three genetically linked MYLK coding SNPs (Pro21His, Ser147Pro, Val261Ala) are associated with risk and severity of acute respiratory distress syndrome (ARDS) and severe asthma

• These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation

• Quantifiable cell immunofluorescence assays have demonstrated that both the Ser147Pro variant alone and the combined three SNP variant show reduced Y464 phosphorylation:

  • At 2 minutes post-S1P stimulation: 20% decrease for S147P-nmMLCK1 and 40% decrease for 3SNP-nmMLCK1 compared to wild-type

  • At 5 minutes post-S1P stimulation: 14% decrease for S147P and 32% decrease for 3SNP variant

This reduced phosphorylation correlates with attenuated nmMLCK translocation to the cell periphery and retarded lamellipodial protrusion, potentially explaining the mechanism by which these SNPs increase susceptibility to inflammatory lung injury .

What is the relationship between S1P signaling, MYLK Y464 phosphorylation, and vascular barrier function?

The relationship between S1P, MYLK Y464 phosphorylation, and barrier function follows a mechanistic cascade:

• S1P (sphingosine 1-phosphate) acts as a potent bioactive endogenous lipid that signals cytoskeletal rearrangement

• S1P stimulation induces critical nmMLCK Y464 and Y471 phosphorylation through c-Abl-mediated pathways

• This phosphorylation results in nmMLCK translocation to the cell periphery and lamellipodia

• At the periphery, spatially-directed increases in myosin light chain (MLC) phosphorylation occur

• Increased MLC phosphorylation generates localized tension that promotes:

  • Lamellipodia protrusion

  • Increased cell-cell adhesion

  • Enhanced vascular barrier integrity

The temporal dynamics are important: Y464 phosphorylation and subsequent translocation coincide exactly with increases in trans-EC electrical resistance (TER) and elastic modulus at the cell periphery (measured via atomic force microscopy) . MYLK SNPs that reduce Y464 phosphorylation functionally influence these barrier-regulatory cytoskeletal responses, potentially explaining their association with inflammatory lung diseases .

What methodologies are most effective for studying MYLK Y464 phosphorylation dynamics?

Several complementary techniques have proven effective for investigating MYLK Y464 phosphorylation:

• Quantifiable cell immunofluorescence assays:

  • Allow visualization and quantification of phosphorylation in situ

  • Can be combined with time-course analysis following S1P stimulation

  • Enable measurement of mean fluorescence intensity per unit area

• Live cell imaging:

  • Permits real-time observation of nmMLCK translocation

  • Can be used to study co-localization with other proteins (e.g., cortactin)

  • Reveals dynamics of membrane ruffling and lamellipodia formation

• Kymographic assays:

  • Provide quantitative analysis of lamellipodial protrusion dynamics

  • Can demonstrate functional consequences of altered phosphorylation

• Atomic force microscopy (AFM):

  • Measures changes in elastic modulus at the cell periphery

  • Provides biophysical correlates to phosphorylation events

• Trans-endothelial electrical resistance (TER):

  • Quantifies barrier integrity changes that correlate with phosphorylation

These techniques are most powerful when used in combination, allowing researchers to correlate molecular events (phosphorylation) with cellular functions (localization, barrier integrity) in real time.

How can researchers distinguish between effects of different MYLK phosphorylation sites?

Distinguishing between the functional effects of Y464 phosphorylation versus other sites (such as Y471) requires several strategic approaches:

• Site-specific phospho-antibodies:

  • Use antibodies that specifically recognize MYLK phosphorylated at Y464

  • Compare with antibodies targeting other phosphorylation sites

• Site-directed mutagenesis:

  • Create Y464F mutants (non-phosphorylatable) to specifically ablate Y464 phosphorylation

  • Generate compound mutants (Y464F/Y471F) to assess additive effects

  • Express these mutants in cells to determine site-specific functions

• Phospho-mimetic mutations:

  • Y464E or Y464D mutations can mimic constitutive phosphorylation

  • Compare phenotypes between phospho-null and phospho-mimetic mutants

• Temporal analysis:

  • Different phosphorylation sites may show distinct kinetics after stimulation

  • Time-course experiments can reveal the sequence of phosphorylation events

• Mass spectrometry:

  • Provides direct detection and quantification of site-specific phosphorylation

  • Can identify novel sites and determine stoichiometry of phosphorylation

Using these approaches in combination provides the most comprehensive understanding of site-specific functions in MYLK regulation.

What are the optimal conditions for detecting MYLK Y464 phosphorylation in different experimental systems?

Optimal detection conditions vary by experimental system and technique:

For immunohistochemistry (IHC):

• Dilution range: 1:100-1:300 of antibody

• Antigen retrieval: Sodium citrate pH 6.0 at >98°C for 20 minutes

• Fixation: Paraformaldehyde fixation preserves phospho-epitopes

• Visualization: Secondary antibody dilution at 1:200 is effective

For Western blotting:

• Sample preparation: Use phosphatase inhibitors during lysis

• Gel conditions: 5-20% SDS-PAGE gel at 70V (stacking)/90V (resolving)

• Transfer: Transfer to nitrocellulose at 150mA for 50-90 minutes

• Blocking: 5% non-fat milk in TBS for 1.5 hours at room temperature

• Primary antibody: 0.5 μg/mL overnight at 4°C

• Secondary antibody: Goat anti-rabbit IgG-HRP at 1:10000 dilution

• Detection: Enhanced chemiluminescence

For cell-based assays:

• Stimulation: S1P treatment shows peak Y464 phosphorylation at 2-5 minutes

• Cell type: Human lung microvascular endothelial cells show robust phosphorylation

• Fixation timing: Rapid fixation is critical to preserve phosphorylation status

• Controls: Include both stimulated and unstimulated samples for comparison

What technical challenges exist in studying nmMLCK translocation and phosphorylation?

Researchers face several technical challenges when investigating nmMLCK phosphorylation and translocation:

• Phosphorylation site specificity:

  • Y464 phosphorylation must be distinguished from other phosphorylation events

  • Antibody specificity must be rigorously validated

• Temporal dynamics:

  • Phosphorylation events are often transient, requiring precise timing

  • Peak phosphorylation occurs at specific time points (2-5 minutes post-S1P stimulation)

• Spatial resolution:

  • Detecting translocation to specific subcellular compartments requires high-resolution imaging

  • Co-localization with cytoskeletal elements demands multi-channel imaging capabilities

• Genetic variation effects:

  • MYLK SNPs introduce heterogeneity in phosphorylation responses

  • Requires careful genetic characterization of experimental systems

• Quantification challenges:

  • Measuring fluorescence intensity requires standardized methods

  • Normalizing for cell size, shape, and total protein expression

• Functional correlation:

  • Connecting phosphorylation events to functional outcomes (barrier integrity, contractility)

  • Requires integration of multiple experimental approaches

Addressing these challenges requires a multi-faceted approach combining biochemical, genetic, and imaging techniques with appropriate controls and standardization.

How can researchers best utilize Phospho-MYLK (Y464) Antibody to investigate vascular permeability mechanisms?

To effectively investigate vascular permeability mechanisms using Phospho-MYLK (Y464) Antibody, researchers should implement the following methodological approach:

• Integrated experimental system:

  • Use human lung microvascular endothelial cells as a model system

  • Establish both in vitro and in vivo systems to validate findings across models

• Combined phosphorylation and functional assays:

  • Correlate Y464 phosphorylation with barrier function measurements (TER)

  • Monitor actin cytoskeleton rearrangement concurrently with phosphorylation events

• Genetic manipulation strategies:

  • Express wild-type versus SNP variants of MYLK to assess functional impacts

  • Use siRNA knockdown with reconstitution of mutant forms for mechanistic studies

• Temporal analysis:

  • Conduct detailed time-course experiments following S1P stimulation

  • Focus on 2-5 minute timepoints where maximal phosphorylation differences are observed

• Spatial analysis:

  • Quantify translocation to cell periphery as a function of phosphorylation

  • Measure co-localization with cytoskeletal markers and adhesion proteins

• Translational considerations:

  • Correlate findings with human genetic data from patients with vascular barrier dysfunction

  • Investigate potential therapeutic approaches to modulate Y464 phosphorylation

This comprehensive approach enables researchers to establish causal relationships between molecular events (Y464 phosphorylation), cellular processes (nmMLCK translocation), and physiological outcomes (vascular barrier integrity).