Phospho-MYL9 (Tyr118) Antibody

What is MYL9 and what cellular functions does it regulate?

MYL9 (myosin light chain 9, regulatory) is a myosin regulatory subunit that plays an essential role in regulating both smooth muscle and nonmuscle cell contractile activity via its phosphorylation. It functions as a structural component of myosin, which consists of two heavy chains and four light chains. MYL9 is implicated in several cellular processes including:

• Regulation of muscle contraction by modulating the ATPase activity of myosin heads

• Cell adhesion, polarity, and motility

• Cytokinesis and receptor capping

• Cell migration and invasion

MYL9 binds calcium and is activated by myosin light chain kinase. The protein has a molecular weight of approximately 20 kDa and is also known by multiple aliases including LC20, MLC-2C, MLC2, MRLC1, and MYRL2 .

What is the significance of MYL9 phosphorylation in cancer research?

MYL9 has emerged as a critical player in cancer biology with varying roles depending on the cancer type:

• In squamous cervical cancer (SCC), MYL9 is upregulated compared to peritumoral tissues, and promotes cancer cell migration and invasion by enhancing aerobic glycolysis through the JAK2/STAT3 pathway .

• In colorectal cancer, MYL9 overexpression promotes cell proliferation, invasion, migration, and angiogenesis by binding to YAP1 and activating Hippo signaling .

• Studies have shown contradictory roles in different cancers - MYL9 exhibits tumor suppression functions in gastric and colon cancers, while demonstrating oncogenic roles in melanoma, glioblastoma, and breast cancer .

This dichotomy makes MYL9 phosphorylation status an important biomarker for potentially targeted treatment approaches.

What experimental techniques can be used with Phospho-MYL9 antibodies?

Phospho-MYL9 antibodies can be utilized in various experimental applications:

The most common applications include Western blotting for quantifying phosphorylation levels and immunofluorescence for visualizing subcellular localization of phosphorylated MYL9 .

How should researchers design experiments to study MYL9 phosphorylation dynamics?

When designing experiments to study MYL9 phosphorylation:

• Include appropriate positive controls: For Tyr118 phosphorylation studies, consider using cell lines like 293T, C2C12, or C6, which have been validated as positive samples .

• Employ temporal analysis: MYL9 phosphorylation states change rapidly in response to stimuli. Design time-course experiments with multiple timepoints (e.g., 0, 5, 15, 30, 60 min) after stimulation.

• Use multiple detection methods: Combine Western blotting with immunofluorescence to correlate total phosphorylation levels with subcellular localization.

• Include pathway inhibitors: When studying signaling mechanisms, incorporate specific inhibitors of upstream kinases to establish causality.

• Consider the matrix/substrate context: The phosphorylation of MYL9 can be affected by the extracellular matrix and tension state of cells .

What are the recommended protocols for detecting phosphorylated MYL9 in Western blots?

For optimal Western blot detection of phosphorylated MYL9:

• Sample preparation:

  • Rapidly lyse cells in buffer containing phosphatase inhibitors to prevent dephosphorylation

  • Maintain samples at 4°C during processing

  • Use SDS-PAGE gels with appropriate resolution for the 20 kDa MYL9 protein

• Transfer and detection:

  • Use PVDF membranes for optimal protein binding

  • Block with 5% BSA in TBST (not milk) as milk contains phosphoproteins that can increase background

  • Dilute primary phospho-specific antibody 1:500-1:1000 in 5% BSA/TBST

  • Include controls with phosphatase-treated lysates to confirm specificity

• Stripping and reprobing:

  • After detecting phospho-MYL9, strip and reprobe for total MYL9 to calculate phosphorylation ratio

  • Use GAPDH as a loading control, as demonstrated in RT-qPCR experiments with MYL9

How can researchers optimize immunofluorescence protocols for phospho-MYL9 detection?

For high-quality immunofluorescence detection of phosphorylated MYL9:

• Fixation methods:

  • 4% paraformaldehyde for 15 minutes at room temperature preserves phospho-epitopes

  • Avoid methanol fixation which can cause loss of phosphorylation signal

• Permeabilization:

  • Use 0.1-0.2% Triton X-100 for 5-10 minutes

  • Alternative: 0.5% saponin for gentler permeabilization

• Blocking and antibody dilution:

  • Block with 5% normal serum from the species of the secondary antibody

  • Dilute phospho-MYL9 primary antibody 1:200-1:800 in blocking buffer

• Co-staining suggestions:

  • Include phalloidin to visualize F-actin structures

  • Consider co-staining for paxillin to identify focal adhesions

• Imaging considerations:

  • Use confocal microscopy for precise subcellular localization

  • Perform Z-stack imaging to capture the full cellular architecture

How does MYL9 phosphorylation contribute to cancer cell migration and invasion?

MYL9 phosphorylation plays a complex role in cancer cell migration and invasion mechanisms:

• In squamous cervical cancer:

  • MYL9 knockdown significantly suppresses cancer cell migration and invasion

  • This effect is mediated through reduced aerobic glycolysis

  • MYL9 promotes JAK2/STAT3 pathway activity, enhancing expression of glycolytic enzymes GLUT1, HK2, and LDHA

• In colorectal cancer:

  • MYL9 overexpression promotes cell invasion and migration

  • This occurs through binding to YAP1 and activating Hippo signaling

  • Migration effects can be measured through wound healing assays

  • Invasion can be quantified using transwell invasion assays

• Mechanistic considerations:

  • MYL9 phosphorylation affects actin-myosin binding and contractility

  • This modulates the formation of stress fibers and focal adhesions

  • The reorganization of the actin cytoskeleton is essential for cell movement

Research comparing different phosphorylation sites has shown that while Thr18/Ser19 phosphorylation primarily regulates contractility, other phosphorylation events, potentially including Tyr118, may have distinct functions in specific cellular contexts.

What are the signaling pathways that regulate MYL9 phosphorylation?

Multiple signaling pathways contribute to MYL9 phosphorylation in different cellular contexts:

• JAK2/STAT3 pathway:

  • Plays a critical role in MYL9-mediated cancer progression

  • MYL9 knockdown reduces phosphorylation of JAK2 and STAT3

  • This pathway connects MYL9 to enhanced aerobic glycolysis in cancer cells

• Hippo signaling pathway:

  • MYL9 binds directly to YAP1 (Yes-associated protein 1)

  • This interaction activates downstream Hippo signaling

  • Affects expression of connective tissue growth factor and cysteine-rich angiogenic inducer 61

• TGFβ pathway:

  • TGFβ stimulation influences myosin light chain phosphorylation

  • This affects cardiac fibroblast matrix synthesis and remodeling

  • Mechanotransduction through matrix tension interacts with this pathway

• RhoA/ROCK pathway:

  • Classical pathway regulating myosin light chain phosphorylation

  • Primarily affects Thr18/Ser19 phosphorylation

  • Contributes to stress fiber formation and cell contractility

Understanding these pathways provides opportunities for targeted interventions in diseases where MYL9 dysregulation contributes to pathology.

What methods can detect changes in MYL9 expression and how do they compare to phosphorylation detection?

Multiple complementary approaches can be used to analyze MYL9 expression and phosphorylation:

RT-qPCR protocol example from SCC research:

• PCR mix: 1 μL cDNA, 1× SYBR Green, specific primers

• Thermocycling: 95°C for 10 min, followed by 45 cycles of 95°C for 15s, 60°C for 15s, 72°C for 10s

• MYL9 primers: forward 5′-GCCACATCCAATGTCTTCGC-3′, reverse 5′-GCGTTGCGAATCACATCCTC-3′

• Normalization to GAPDH using the 2−ΔΔCt method

What are common issues with phospho-specific antibodies and how can they be addressed?

Researchers frequently encounter these challenges when working with phospho-specific antibodies:

• Loss of phosphorylation signal:

  • Ensure samples are processed quickly with phosphatase inhibitors

  • Avoid freeze-thaw cycles of lysates

  • Store antibodies according to manufacturer recommendations (typically -20°C, avoid freeze/thaw cycles)

• Cross-reactivity concerns:

  • Validate antibody specificity with phosphatase-treated controls

  • Consider using knockout/knockdown samples as negative controls

  • Verify results with multiple antibody clones when possible

• Background issues:

  • Use BSA rather than milk for blocking phospho-specific antibodies

  • Optimize primary antibody concentration (start with 1:1000 for WB, 1:200 for IF)

  • Include phospho-blocking peptides as controls

• Inconsistent results between experiments:

  • Standardize cell culture conditions that affect phosphorylation status

  • Control timing precisely between sample collection and lysis

  • Use positive control samples with known phosphorylation status

How can researchers distinguish between different phosphorylation sites on MYL9?

Distinguishing between different phosphorylation sites requires careful experimental design:

• Antibody selection:

  • Use site-specific antibodies targeting distinct phosphorylation sites (e.g., separate antibodies for Thr18/Ser19 vs. Tyr118)

  • Verify antibody specificity with blocking peptides containing the phosphorylated residue

• Validation approaches:

  • Mutational analysis: Create site-specific mutants (e.g., Y118F) to prevent phosphorylation at specific sites

  • Phosphatase treatments: Use site-specific phosphatases when available

  • Mass spectrometry: For definitive identification of all phosphorylation sites

• Kinase inhibition:

  • Target different upstream kinases that preferentially phosphorylate specific sites

  • Time-course studies to detect sequential phosphorylation events

• Functional assays:

  • Compare phenotypic effects of blocking different phosphorylation sites

  • Assess interactions with binding partners that may be phosphorylation site-specific

What positive and negative controls should be included when working with phospho-MYL9 antibodies?

A robust experimental design should include these controls:

Positive controls:

• Cell lines known to express phosphorylated MYL9 (e.g., 293T, C2C12, C6)

• Tissues with high MYL9 expression (e.g., smooth muscle, bladder, colon)

• Stimulated samples: Cells treated with factors known to induce phosphorylation

Negative controls:

• Phosphatase-treated samples to remove phosphorylation

• Blocking peptide competition to demonstrate antibody specificity

• MYL9 knockdown/knockout samples where available

• Appropriate isotype control antibodies matching the primary antibody host species

Technical controls:

• Total MYL9 antibody staining to normalize phosphorylation levels

• Housekeeping protein controls (e.g., GAPDH for Western blots)

• Secondary antibody-only controls to assess non-specific binding