Phospho-MYL9 (Ser19) Antibody

What is MYL9 and what is the significance of its phosphorylation at Ser19?

Myosin regulatory light polypeptide 9 (MYL9), also known as MLC2, is a regulatory subunit of myosin that plays a crucial role in both smooth muscle and nonmuscle cell contractile activity. The protein has a molecular weight of approximately 19-20 kDa and consists of 172 amino acid residues in humans .

Phosphorylation at Ser19 (sometimes designated as Ser20 when including the initiator methionine) is particularly significant because:

• It activates myosin ATPase activity, which directly correlates with smooth muscle contraction

• It regulates the assembly of stress fibers in nonmuscle cells

• It is essential for cytoskeletal reorganization during processes like cytokinesis, receptor capping, and cell locomotion

This phosphorylation is primarily mediated by Ca²⁺/calmodulin-dependent myosin light chain kinases and ROCK (Rho-associated protein kinase) .

What are the technical considerations for validating Phospho-MYL9 (Ser19) antibody specificity?

When validating the specificity of Phospho-MYL9 (Ser19) antibodies, researchers should implement several strategies:

• Phosphatase treatment controls: Use λ phosphatase-treated lysates as negative controls to confirm phospho-specificity

• Phosphorylation induction: Treat cells with phosphorylation inducers like calyculin A to generate positive controls with increased phospho-MYL9 levels

• Cross-reactivity testing:

  Tested Reactivity	Common Reactive Species
  Confirmed	Human, mouse, rat
  Predicted	Other mammalian species based on sequence homology

• Western blot validation: Look for a single band at the expected molecular weight (18-20 kDa)

• Immunofluorescence pattern: Verify appropriate subcellular localization, typically showing stress fiber-associated pattern in stimulated cells

What are the optimal protocols for detecting Phospho-MYL9 (Ser19) using Western blotting?

For optimal Western blot detection of Phospho-MYL9 (Ser19), follow these methodological considerations:

• Sample preparation:

  • Rapidly harvest cells in phosphatase inhibitor-containing buffer to preserve phosphorylation status

  • For enhanced detection, treat cells with calyculin A prior to harvesting

• Antibody dilutions:

  Antibody Source	Recommended Dilution
  Cell Signaling (#3671)	1:1000
  Proteintech (82717-1-RR)	1:5000-1:50000
  Proteintech (29504-1-AP)	1:500-1:2000

• Visualization protocol:

  • Use 12-15% SDS-PAGE gels for optimal resolution of the 18-20 kDa band

  • Transfer to PVDF or nitrocellulose membrane

  • Block with 5% BSA (preferred over milk for phospho-epitopes)

  • Incubate with primary antibody overnight at 4°C

  • Detection using appropriate secondary antibody and chemiluminescence system

• Technical considerations:

  • Always run phosphatase-treated negative controls

  • Include loading controls that are not affected by your experimental conditions

How can Phospho-MYL9 (Ser19) antibodies be optimized for flow cytometry applications?

For flow cytometry detection of phospho-MYL9, consider these methodological approaches:

• Sample preparation:

  • Fix cells with 4% formaldehyde

  • Permeabilize with methanol or 0.2% Triton X-100

  • Use single-cell suspensions at approximately 10^6 cells per 100 μl

• Antibody concentration:

  • Use 0.25 μg antibody per 10^6 cells in a 100 μl suspension

  • For Proteintech antibody 29504-1-AP, use 0.13 μg per 10^6 cells

• Controls required:

  • Unstained cells

  • Isotype control (Rabbit IgG)

  • Phosphatase-treated negative control cells

  • Calyculin A-treated positive control cells

• Gating strategy:

  • Gate on viable cells first

  • Analyze phospho-MYL9 signal intensity as compared to baseline controls

How does Junb regulate MYL9 phosphorylation and what are the implications for cardiovascular research?

Research has revealed an important regulatory relationship between the transcription factor Junb and MYL9 that has significant implications for cardiovascular research:

• Transcriptional regulation:

  • Junb directly binds to the MYL9 promoter region at CRE sites located at positions -1,688 and -1,666 upstream of the transcriptional start site

  • ChIP analysis confirms Junb occupancy of this promoter region, making MYL9 a direct Junb target gene

• Functional implications:

  • Junb-deficient vessels show reduced MYL9 expression and phosphorylation

  • This leads to decreased arterial contractile capacity

  • Junb-deficient mice fail to develop hypertension in response to DOCA-salt treatment

• Mechanistic pathway:

  Pathway Component	Function	Effect of Junb Deficiency
  Junb	Transcription factor	Reduced expression
  MYL9	Regulatory light chain	Decreased expression
  Phospho-MYL9 (Ser19)	Active form	Severely reduced
  Arterial contraction	Physiological response	Impaired

• Research applications:

  • Phospho-MYL9 (Ser19) detection can serve as a biomarker for vascular smooth muscle contractility

  • Potential therapeutic target for hypertension and other cardiovascular disorders

What experimental design considerations are important when investigating Phospho-MYL9 (Ser19) in cellular contractility studies?

When designing experiments to study the role of Phospho-MYL9 (Ser19) in cellular contractility, researchers should consider:

• Stimulation methods:

  • Pharmacological agents: Use norepinephrine (10^-8 to 10^-5 M) for smooth muscle contraction

  • Mechanical stimulation: Apply cyclic stretch to isolated arteries

• Inhibitor studies:

  • ROCK inhibitors (Y-27632) to block Ser19 phosphorylation

  • MLCK inhibitors (ML-7) to differentiate between kinase pathways

  • Combined inhibition to assess pathway redundancy

• Quantification approaches:

  Measurement	Technique	Parameter
  Contractility	Isolated vessel perfusion	Vessel diameter changes
  Phosphorylation levels	Western blotting	Band intensity ratio
  Cellular mechanics	Traction force microscopy	Cell-generated forces
  Cytoskeletal dynamics	Immunofluorescence	Stress fiber formation

• Model systems comparison:

  • Isolated vessels (maintain tissue architecture)

  • Primary VSMCs (cell-type specificity)

  • Cell lines (experimental convenience)

  • Genetic models (Junb knockout or MYL9 mutations)

How can phospho-specific antibodies be used to distinguish between single and dual phosphorylation states of MYL9?

MYL9 can be phosphorylated at both Thr18 and Ser19 sites (Thr19/Ser20 when including the initiator methionine), creating a technical challenge in distinguishing phosphorylation states. Here's a methodological approach:

• Antibody selection strategy:

  Phosphorylation State	Recommended Antibody	Detection Method
  Single (pSer19 only)	Anti-Phospho-MYL9 (Ser19)	WB, IF
  Dual (pThr18/pSer19)	Anti-Phospho-MYL9 (Thr18/Ser19)	WB, IF
  Total MYL9	Anti-MYL9 (non-phospho-specific)	WB, IF

• Analytical approaches:

  • Sequential immunoprecipitation with single-site antibodies followed by detection with dual-site antibodies

  • 2D gel electrophoresis to separate different phospho-forms based on charge

  • Phosphatase treatment followed by in vitro kinase assays with site-specific kinases

• Mass spectrometry validation:

  • For definitive identification of phosphorylation sites

  • Can provide relative quantification of single vs. dual phosphorylated forms

• Biological significance:

  • Single phosphorylation (Ser19) is sufficient to activate myosin ATPase

  • Dual phosphorylation (Thr18/Ser19) further enhances ATPase activity and stabilizes myosin filaments

What are common pitfalls when detecting Phospho-MYL9 (Ser19) and how can they be addressed?

Researchers frequently encounter these challenges when working with Phospho-MYL9 (Ser19) antibodies:

• Loss of phosphorylation signal:

  • Cause: Phosphatase activity during sample preparation

  • Solution: Add phosphatase inhibitors immediately; keep samples cold; use calyculin A pre-treatment

• Cross-reactivity with related proteins:

  • Cause: Sequence similarity with other myosin light chains (MYL12A/MYL12B)

  • Solution: Validate with knockout/knockdown controls; confirm molecular weight (19-20 kDa for MYL9)

• Inconsistent immunofluorescence staining:

  • Cause: Fixation methods affecting epitope accessibility

  • Solution: Optimize fixation protocol; use 4% formaldehyde followed by Triton X-100 permeabilization

• Variable baseline phosphorylation:

  • Cause: Cell culture conditions affecting basal contractility

  • Solution: Standardize culture conditions; use serum starvation before experiments; include appropriate controls

How can researchers optimize detection conditions for weak Phospho-MYL9 (Ser19) signals?

For enhanced detection of weak Phospho-MYL9 (Ser19) signals, consider these methodological improvements:

• Signal amplification strategies:

  • Use highly sensitive detection systems (enhanced chemiluminescence)

  • Consider tyramide signal amplification for immunofluorescence

  • Try biotin-streptavidin systems for additional sensitivity

• Sample enrichment approaches:

  • Immunoprecipitation before Western blotting

  • Phospho-protein enrichment columns

  • Subcellular fractionation to concentrate cytoskeletal components

• Antibody optimization:

  Parameter	Optimization Strategy
  Incubation time	Extend to overnight at 4°C
  Antibody concentration	Titrate to identify optimal concentration
  Blocking agent	Use 5% BSA instead of milk for phospho-epitopes
  Secondary antibody	Select high-sensitivity detection systems

• Physiological stimulation:

  • Use ROCK activators or calcium ionophores to increase phosphorylation levels

  • Calyculin A treatment (phosphatase inhibitor) can increase phospho-MYL9 signal