Phospho-MYL9 (Ser15) Antibody

What is Phospho-MYL9 (Ser15) Antibody and what cellular functions does it help investigate?

Phospho-MYL9 (Ser15) Antibody specifically detects endogenous levels of MYL9 (Myosin Light Chain 2) only when phosphorylated at serine 15 . This antibody serves as a critical research tool for investigating cellular contractility, migration, and signaling pathways. MYL9 functions as a myosin regulatory subunit that plays an important role in regulating both smooth muscle and nonmuscle cell contractile activity via its phosphorylation . It is implicated in essential cellular processes including cytokinesis, receptor capping, and cell locomotion . The antibody enables researchers to specifically track the phosphorylated state of MYL9, which represents its activated form in various cellular contexts.

Which experimental applications are most suitable for Phospho-MYL9 (Ser15) Antibody?

The Phospho-MYL9 (Ser15) Antibody has been validated for multiple experimental applications:

The antibody's high specificity makes it particularly valuable for studying phosphorylation events in signaling cascades related to cellular contractility and migration . When designing experiments, researchers should consider the specific cellular context and select the appropriate application based on whether protein quantification, localization, or interaction studies are needed.

How should Phospho-MYL9 (Ser15) Antibody be stored to maintain optimal activity?

To preserve antibody activity and specificity, Phospho-MYL9 (Ser15) Antibody should be stored at -20°C or -80°C upon receipt . Repeated freeze-thaw cycles should be avoided as they can significantly degrade antibody performance . The antibody is typically supplied in phosphate buffered saline (without Mg²⁺ and Ca²⁺), pH 7.4, 150mM NaCl, 0.02% sodium azide, and 50% glycerol . This formulation helps maintain stability during storage. For laboratories conducting long-term studies, aliquoting the antibody into single-use volumes before freezing is recommended to prevent degradation from multiple freeze-thaw cycles.

How can researchers optimize Western blot protocols specifically for Phospho-MYL9 (Ser15) detection?

Optimizing Western blot protocols for Phospho-MYL9 (Ser15) detection requires several critical considerations:

• Sample preparation: Include phosphatase inhibitors in lysis buffers to preserve phosphorylation status. The protocol used in recent MYL9 research demonstrates effectiveness: "Flasks containing cells were washed three times with ice-cold phosphate-buffered saline, and total protein was harvested using RIPA lysis buffer containing phenylmethanesulfonyl fluoride and phosphatase inhibitors at a ratio of 100:1:1" .

• Gel selection: Use 10-12% polyacrylamide gels for optimal resolution of the 19-20 kDa MYL9 protein .

• Blocking conditions: Block membranes with 5% bovine serum albumin in TBST (not milk, which contains phosphatases) for 1 hour at 37°C .

• Antibody dilution: Use the Phospho-MYL9 (Ser15) Antibody at 1:500-1:1000 dilution for optimal signal-to-noise ratio .

• Positive controls: Include samples treated with phosphatase activators (e.g., calyculin A) as positive controls .

• Validation approach: Consider using lambda phosphatase-treated samples as negative controls to confirm phospho-specificity .

This methodological approach ensures specific detection of the phosphorylated form while minimizing background and non-specific signals.

What are the key considerations when designing immunofluorescence experiments with Phospho-MYL9 (Ser15) Antibody?

When designing immunofluorescence experiments with Phospho-MYL9 (Ser15) Antibody, researchers should address these critical factors:

• Fixation method: Methanol fixation has been successfully used for immunofluorescence staining with phospho-specific MYL9 antibodies, as demonstrated in published protocols . Paraformaldehyde fixation (4%) followed by permeabilization with 0.1% Triton X-100 is an alternative approach.

• Antibody dilution: Use at 1:100-1:200 dilution for optimal staining with minimal background .

• Blocking strategy: Block with 5% normal serum from the same species as the secondary antibody to reduce non-specific binding.

• Controls: Include both phosphatase-treated negative controls and phosphorylation-stimulated positive controls.

• Co-localization studies: Consider dual staining with markers of contractile structures (e.g., F-actin) or specific cellular compartments to provide context for phospho-MYL9 localization.

• Cell types: U87 cells have been validated for immunofluorescence with this antibody , but optimization may be required for other cell types.

These considerations help ensure reliable subcellular localization data for phosphorylated MYL9 while minimizing artifacts.

How can researchers effectively validate the specificity of Phospho-MYL9 (Ser15) Antibody in their experimental system?

Validating antibody specificity is crucial for meaningful results. For Phospho-MYL9 (Ser15) Antibody, implement these validation strategies:

• Phosphatase treatment: Treat positive samples with lambda phosphatase to demonstrate loss of signal due to dephosphorylation .

• Stimulation experiments: Compare basal versus stimulated conditions known to induce MYL9 phosphorylation (e.g., thrombin treatment for endothelial cells).

• Peptide competition: Pre-incubate the antibody with phosphorylated and non-phosphorylated peptides to confirm phospho-specificity.

• siRNA validation: Use MYL9 siRNA knockdown to demonstrate reduced signal, confirming target specificity.

• Multiple detection methods: Validate findings using orthogonal methods such as mass spectrometry or alternative phospho-specific antibodies targeting different epitopes.

• Cross-species reactivity: The antibody is designed for human MYL9 detection , so validation is essential when using with other species.

The manufacturer's purification method supports specificity: "Antibodies were purified by affinity-chromatography using epitope-specific phosphopeptide. Non-phospho specific antibodies were removed by chromatography using non-phosphopeptide" .

How has Phospho-MYL9 (Ser15) Antibody been used to investigate cancer cell migration and invasion?

Research using phospho-specific MYL9 antibodies has revealed important roles in cancer progression:

• Squamous cervical cancer (SCC): Recent studies demonstrated that MYL9 is upregulated in SCC tissues compared to peritumoral samples, promoting migration and invasion . Specifically, researchers found that "MYL9 knockdown inhibited the migration and invasion of SCC cells by regulating aerobic glycolysis and its downstream factors (including GLUT1, HK2, and LDHA) via the JAK2/STAT3 pathway" .

• Experimental approaches: Transwell and Boyden assays were used to assess the role of MYL9 in cancer cell migration and invasion . Western blotting with phospho-specific antibodies helped establish the connection between MYL9 phosphorylation status and invasive phenotypes.

• Mechanism elucidation: Phospho-MYL9 antibodies enabled researchers to demonstrate that "MYL9 induces JAK2/STAT3 pathway activity and promotes SCC migration and invasion by enhancing aerobic glycolysis" .

• Differential expression: The table below summarizes MYL9 mRNA expression across 36 SCC patients, highlighting its potential as a biomarker:

These findings suggest that phosphorylated MYL9 serves as a potential metastasis-related biomarker in cancer research .

What methodological approaches can researchers use to study the relationship between MYL9 phosphorylation and aerobic glycolysis?

To investigate the relationship between MYL9 phosphorylation and aerobic glycolysis, researchers can employ these methodological approaches:

• Lactate measurement: Quantify lactate levels in control versus MYL9-knockdown cells to assess glycolytic output. Recent research demonstrated that "lactate levels were lower in the siMYL9 group" .

• Expression analysis of glycolytic enzymes: Perform Western blotting to detect key glycolytic enzymes such as GLUT1, HK2, and LDHA. Studies showed that "GLUT1, HK2, and LDHA protein expression was significantly inhibited after MYL9 knockdown" .

• Signaling pathway investigation: Examine phosphorylation status of potential mediators such as JAK2/STAT3 pathway components in relation to MYL9 phosphorylation. Research indicated that "phosphorylation of JAK2 and STAT3 was significantly reduced in the siMYL9 group" .

• Functional rescue experiments: Perform rescue experiments with constitutively active pathway components to establish causal relationships between MYL9 phosphorylation and metabolic reprogramming.

• Metabolic flux analysis: Use isotope-labeled glucose to track metabolic pathways and determine how MYL9 phosphorylation affects glucose utilization.

These approaches collectively provide a comprehensive framework for investigating how phosphorylated MYL9 regulates metabolic processes in cancer and other cellular contexts.

What are common technical challenges when using Phospho-MYL9 (Ser15) Antibody and how can they be addressed?

Researchers may encounter several technical challenges when working with Phospho-MYL9 (Ser15) Antibody:

• Loss of phosphorylation signal:

  • Problem: Phosphorylation sites are often labile during sample processing.

  • Solution: Always include phosphatase inhibitors in lysis buffers and maintain samples at 4°C during processing . Use fresh samples whenever possible.

• Cross-reactivity issues:

  • Problem: Potential cross-reactivity with other phosphorylated myosin light chains.

  • Solution: Validate specificity using knockout/knockdown controls or lambda phosphatase treatment . The antibody is purified to remove non-phospho specific antibodies through chromatography .

• Variable phosphorylation levels:

  • Problem: Phosphorylation status can vary depending on cell culture conditions.

  • Solution: Standardize cell culture conditions and serum starvation protocols. Consider using phosphatase inhibitors like calyculin A as positive controls .

• Background in immunofluorescence:

  • Problem: High background staining in IF applications.

  • Solution: Optimize antibody concentration (1:100-1:200 is recommended ), improve blocking (5% BSA), and ensure thorough washing steps.

• Multiple bands in Western blots:

  • Problem: Detection of additional bands besides the expected 19-20 kDa band.

  • Solution: Optimize SDS-PAGE conditions, ensure complete sample denaturation, and consider using gradient gels for better resolution.

Addressing these challenges systematically will improve experimental outcomes with phospho-specific antibodies.

How should researchers interpret differences in phosphorylation patterns detected across different phospho-specific MYL9 antibodies?

When working with different phospho-specific MYL9 antibodies (e.g., Ser15, Ser18/19, Thr18/Ser19), researchers should consider these interpretive guidelines:

• Nomenclature differences: First, recognize that phosphorylation site numbering may vary between antibodies. For example, some sources refer to Ser15, while others refer to Ser18 or Ser19 for similar positions due to differences in protein isoforms or whether the initiator methionine is included in the count . The note "This antibody, also known as Thr18/Ser19, is the site where the initiator methionine is removed" exemplifies this issue.

• Functional significance: Different phosphorylation sites have distinct functional implications:

  • Ser15/18 phosphorylation may regulate different aspects of myosin function compared to Thr18/Ser19 phosphorylation

  • Dual phosphorylation (e.g., Thr18+Ser19) often indicates maximal activation

• Kinase specificity: Different kinases target specific phosphorylation sites:

  • MLCK (Myosin Light Chain Kinase) primarily phosphorylates Ser19

  • ROCK (Rho-associated protein kinase) can phosphorylate both Thr18 and Ser19

  • Understanding which kinase is active in your system helps interpret phosphorylation patterns

• Temporal dynamics: Consider that different sites may be phosphorylated with different kinetics:

  • Primary phosphorylation may occur rapidly

  • Secondary sites may show delayed phosphorylation

  • Temporal sampling is crucial for comprehensive understanding

• Validation approach: When discrepancies arise between antibodies recognizing different phospho-sites, validate findings using:

  • Site-specific mutants (S→A or T→A)

  • Phosphatase treatments

  • Kinase inhibitor studies

  • Mass spectrometry to confirm actual phosphorylation sites

Comprehensive analysis using multiple phospho-specific antibodies provides a more complete picture of MYL9 regulation in cellular processes.

How can phospho-MYL9 studies contribute to understanding the JAK2/STAT3 signaling pathway in cancer progression?

Recent research has uncovered important connections between phosphorylated MYL9 and the JAK2/STAT3 pathway in cancer:

• Mechanistic link: Studies demonstrated that "MYL9 induces JAK2/STAT3 pathway activity and promotes SCC migration and invasion by enhancing aerobic glycolysis" . This suggests phosphorylated MYL9 functions upstream of JAK2/STAT3 activation.

• Experimental approach: Western blotting with phospho-specific antibodies revealed that "phosphorylation of JAK2 and STAT3 was significantly reduced in the siMYL9 group" , establishing a direct correlation between MYL9 expression and JAK2/STAT3 pathway activation.

• Research methodology: To further investigate this relationship, researchers can:

  • Use phospho-specific antibodies for MYL9 and JAK2/STAT3 pathway components in parallel

  • Perform co-immunoprecipitation experiments to identify physical interactions

  • Employ kinase inhibitors specific to the JAK2/STAT3 pathway to assess effects on MYL9 phosphorylation

  • Utilize phosphomimetic (S→D) and phosphodeficient (S→A) MYL9 mutants to determine pathway effects

• Translational implications: Understanding this signaling axis provides potential therapeutic targets, as "MYL9 serves as a metastasis-related gene in SCC, and it is a potential biomarker for targeted treatment" .

• Broader oncogenic connections: Research has shown varying roles of MYL9 across cancer types: "MYL9 is weakly expressed in non-small cell lung, gastric, bladder, colon, and prostate cancers" while "MYL9 is highly expressed in ovarian cancer, esophageal squamous cell carcinoma, and glioblastoma" . These differences suggest context-dependent relationships with the JAK2/STAT3 pathway.

Investigating these connections requires integrating phospho-specific detection methods with pathway analysis tools to fully elucidate the regulatory mechanisms.

What methodological considerations are important when studying MYL9 phosphorylation in different cellular contexts and tissue types?

When investigating MYL9 phosphorylation across different cellular contexts and tissue types, researchers should consider these methodological factors:

• Baseline phosphorylation levels: Different tissues exhibit varying basal levels of MYL9 phosphorylation:

  • Smooth muscle tissues generally show higher basal phosphorylation

  • Cancer cells may exhibit dysregulated phosphorylation patterns

  • Standardize sample collection and processing to capture physiological phosphorylation status

• Tissue-specific kinase expression: Consider the expression profiles of relevant kinases:

  • MLCK expression varies across tissues

  • ROCK activity differs between cell types

  • Design experiments that account for the specific kinase environment of your tissue model

• Stimulus response variability: Different tissues respond differently to stimuli affecting MYL9 phosphorylation:

  • Contractile tissues (e.g., smooth muscle) show rapid phosphorylation responses

  • Cancer cells may exhibit constitutive phosphorylation

  • Optimize stimulation protocols for each tissue type

• Cross-species considerations: When working across species:

  • Verify antibody reactivity (human, mouse, rat reactivity has been confirmed for some antibodies)

  • Account for possible phosphorylation site differences between species (e.g., "PhosphorylationH:S18 M:S20 R:S19")

  • Consider using species-specific positive controls

• Sample preparation optimization: Different tissues require adapted protocols:

  • Fibrous tissues may need enhanced homogenization

  • Samples rich in phosphatases may require additional phosphatase inhibitors

  • Fresh frozen versus fixed tissues require different processing approaches

• Validation strategy: Employ tissue-appropriate validation:

  • Use tissue-specific knockdown models

  • Include tissue-matched controls

  • Consider tissue-specific phosphorylation dynamics in experiment design

These methodological considerations ensure reliable and physiologically relevant data on MYL9 phosphorylation across diverse experimental systems.

What emerging technologies can enhance detection and functional analysis of phosphorylated MYL9?

Several cutting-edge technologies are poised to advance phospho-MYL9 research:

• Proximity labeling techniques: BioID or TurboID fused to MYL9 can identify proteins that interact specifically with phosphorylated versus non-phosphorylated forms, revealing phosphorylation-dependent interaction networks.

• Live-cell phosphorylation sensors: Genetically encoded FRET-based sensors for MYL9 phosphorylation enable real-time visualization of phosphorylation dynamics in living cells, providing temporal and spatial resolution not possible with antibody-based detection.

• Single-cell phosphoproteomics: Emerging single-cell phosphoproteomic techniques can reveal cell-to-cell heterogeneity in MYL9 phosphorylation states within tissues or tumors, potentially identifying rare cell populations with distinct signaling profiles.

• CRISPR-based phosphosite editing: Precise genome editing to create phosphomimetic or phosphodeficient mutations at endogenous MYL9 loci provides more physiologically relevant models than overexpression systems.

• Spatial transcriptomics combined with phospho-protein detection: Correlating MYL9 phosphorylation patterns with spatial gene expression profiles in tissue sections can reveal microenvironmental factors regulating phosphorylation.

• Computational modeling: Integration of phosphorylation data into mechanistic models of cytoskeletal dynamics or metabolic networks can predict emergent properties and generate testable hypotheses about MYL9 function.

These technologies will enable researchers to move beyond descriptive studies toward mechanistic understanding of MYL9 phosphorylation in complex biological contexts.

How might understanding MYL9 phosphorylation patterns contribute to therapeutic development for cancer or other diseases?

The translational potential of phospho-MYL9 research spans several therapeutic avenues:

• Biomarker development: Phosphorylated MYL9 shows promise as a diagnostic or prognostic biomarker based on differential expression in cancer tissues. Research has demonstrated that "MYL9 is upregulated in SCC tissues compared with that in peritumoral samples" , suggesting its utility in cancer detection.

• Target identification: Understanding the role of MYL9 phosphorylation in disease progression reveals potential therapeutic targets. Research showing that "MYL9 knockdown inhibited the migration and invasion of SCC cells" indicates that targeting MYL9 or its phosphorylation could impair cancer metastasis.

• Rational drug design: Structural knowledge of phosphorylated MYL9 and its interactions can guide development of:

  • Small molecule inhibitors of MYL9 phosphorylation

  • Disruptors of phospho-MYL9 protein-protein interactions

  • Degraders targeting phosphorylated MYL9 specifically

• Combination therapy approaches: Understanding how MYL9 phosphorylation interfaces with other pathways suggests rational combinations. For example, the finding that "MYL9 promotes SCC cell migration and invasion by enhancing aerobic glycolysis" suggests combining MYL9-targeted therapies with glycolysis inhibitors.

• Patient stratification: Differential expression patterns across patients (as shown in the patient data table ) could identify subgroups likely to respond to specific therapies, enabling personalized medicine approaches.

• Mechanism-based toxicity prediction: Understanding normal physiological roles of phosphorylated MYL9 helps anticipate potential side effects of targeting this pathway, allowing for development of safer therapeutic approaches.