MYL9 Antibody, FITC conjugated

Basic Research Questions

• What is MYL9 and what are its key cellular functions?

  MYL9 (Myosin Light Chain 9) is a myosin regulatory subunit that plays a crucial role in regulating both smooth muscle and nonmuscle cell contractile activity through its phosphorylation state. It functions primarily in the cytoplasm and is implicated in fundamental cellular processes including cytokinesis, receptor capping, and cell locomotion . The protein is also known by several synonyms including LC2, MLC2, MRLC1, MYRL2, and MLC-2C . MYL9 acts as a regulatory component of the myosin complex, and its phosphorylation state dictates contractile force generation in cellular systems. Recent research has expanded our understanding of MYL9 beyond basic contractile functions, revealing its involvement in cancer pathways and inflammatory conditions .

• How does MYL9 expression change in pathological conditions?

  MYL9 expression exhibits disease-specific patterns that may serve as valuable biomarkers. In non-small-cell lung cancer (NSCLC), MYL9 is significantly downregulated both in vivo and in cell cultures compared to normal tissues, suggesting a tumor-suppressive role in this context . This downregulation is associated with increased epithelial-mesenchymal transition (EMT), a key process in cancer progression and metastasis. Conversely, in inflammatory conditions such as Kawasaki disease (KD), plasma MYL9 levels are significantly elevated during the acute phase compared with healthy controls or patients with other febrile illnesses . These levels decline following successful intravenous immunoglobulin (IVIG) therapy in responders but remain elevated in non-responders, indicating that MYL9 may serve as a biomarker for inflammation severity and treatment response . These contrasting expression patterns highlight the context-dependent nature of MYL9 function in different pathologies.

• What is the significance of phosphorylation in MYL9 function?

  Phosphorylation of MYL9, particularly at the Thr19 site, serves as a critical regulatory mechanism for its function. This post-translational modification fundamentally alters MYL9's activity and interaction capabilities within the cell. When phosphorylated, MYL9 enables myosin activation, promoting actin-myosin interaction and subsequent cellular contraction . The phosphorylation state is dynamically regulated by the opposing activities of myosin light chain kinases and phosphatases, allowing for precise temporal and spatial control of contractile events. In research applications, phospho-specific antibodies targeting sites like Thr19 enable investigators to specifically track the active form of MYL9, providing insights into the activation status of myosin-dependent pathways in different cellular contexts . This is particularly valuable when investigating cellular processes where contractility plays a key role, such as cell migration, division, and shape changes associated with EMT in cancer progression.

• How does MYL9 interact with other proteins in cellular signaling pathways?

  MYL9 engages in specific protein-protein interactions that mediate its regulatory functions in cellular signaling. A significant interaction partner identified in recent research is MYO19 (myosin 19). Studies using coimmunoprecipitation and glutathione-S-transferase (GST) pull-down assays have confirmed direct binding between MYL9 and MYO19 . This interaction has functional consequences, particularly in non-small-cell lung cancer (NSCLC), where MYL9 binding to MYO19 suppresses epithelial-mesenchymal transition (EMT). Interestingly, MYO19 expression is significantly increased in NSCLC, with higher levels correlating with worse prognosis, in direct contrast to MYL9's expression pattern . The interaction appears to be regulatory, as MYL9 overexpression reduces MYO19 protein levels, suggesting a potential mechanism for MYL9's tumor-suppressive effects. Through the BioGRID database analysis, researchers have identified additional potential binding partners of MYL9, indicating its involvement in multiple cellular signaling networks beyond simple contractile regulation .

Advanced Technical Questions

• What are the optimal fixation and permeabilization methods for MYL9 immunofluorescence staining?

  For optimal MYL9 immunofluorescence staining, fixation and permeabilization protocols must preserve both protein antigenicity and cellular architecture. Paraformaldehyde fixation (4%) for 15-20 minutes at room temperature is generally recommended for preserving MYL9 epitopes, particularly when using phospho-specific antibodies targeting the Thr19 site. This approach effectively cross-links proteins while maintaining phosphoepitope integrity. For membrane permeabilization, a gentle approach using 0.2-0.3% Triton X-100 for 5-10 minutes typically provides sufficient access to cytoplasmic MYL9 without excessive protein extraction. When working with the FITC-conjugated MYL9 antibodies, it's important to note that these preparations have been validated for various immunofluorescence applications including IF(IHC-P), IF(IHC-F), and IF(ICC) , with recommended dilutions ranging from 1:50-200 . For formalin-fixed paraffin-embedded (FFPE) tissues, heat-induced epitope retrieval using citrate buffer (pH 6.0) is typically required prior to antibody incubation to unmask antigenic sites altered during fixation and embedding processes.

• How should FITC-conjugated MYL9 antibodies be validated for research applications?

  Comprehensive validation of FITC-conjugated MYL9 antibodies is essential before implementing them in critical research applications. A multi-faceted validation approach should include: (1) Specificity verification through Western blotting with positive and negative control lysates to confirm the antibody recognizes a single band of the expected molecular weight (~20 kDa for MYL9); (2) Phospho-specificity validation (for phospho-MYL9 antibodies) using paired samples with and without phosphatase treatment; (3) Immunofluorescence controls including peptide competition assays where pre-incubation with the immunizing peptide should abolish specific staining; (4) Knockdown/knockout validation where cells with genetically reduced MYL9 expression should show corresponding reduction in signal intensity; and (5) Cross-reactivity assessment across species when working with models other than the antibody's primary target species (human, mouse, and rat reactivity has been confirmed for commercial antibodies ). Additionally, researchers should verify the conjugation quality by evaluating the fluorophore-to-protein ratio using spectrophotometric methods and confirming that FITC conjugation hasn't compromised antibody binding through parallel experiments with unconjugated versions of the same antibody clone.

• What are the considerations for using MYL9 antibodies in multiplex immunofluorescence?

  When designing multiplex immunofluorescence experiments incorporating FITC-conjugated MYL9 antibodies, several technical considerations must be addressed. First, spectral compatibility is crucial—FITC (excitation ~495nm, emission ~519nm) must be carefully paired with fluorophores that have minimal spectral overlap to prevent bleed-through. Second, antibody source species must be considered; the rabbit-derived MYL9 polyclonal antibodies should be combined with antibodies raised in different species (mouse, goat, etc.) to prevent cross-reactivity between secondary detection reagents. Third, staining sequence optimization may be necessary—particularly when combining phospho-specific MYL9 detection with other targets, as some epitopes may be more sensitive to certain fixation or permeabilization conditions. Fourth, titration of each antibody in the multiplex panel is essential, as optimal concentrations may differ from those used in single-staining experiments. For studying MYL9's role in EMT, co-staining with markers like E-cadherin, vimentin, or EpCAM would be valuable based on research showing MYL9's regulatory effect on these pathways . Finally, appropriate controls including single-color controls, fluorescence-minus-one (FMO) controls, and isotype controls are mandatory to establish staining specificity in the multiplex context.

• How can researchers optimize signal-to-noise ratio when using FITC-conjugated MYL9 antibodies?

  Optimizing signal-to-noise ratio with FITC-conjugated MYL9 antibodies requires attention to several methodological details. First, antibody titration is essential—while manufacturers recommend dilutions of 1:50-200 for immunofluorescence applications , the optimal concentration should be determined empirically for each tissue or cell type. Second, blocking protocols should be robust, using 5-10% normal serum (from a species different from the primary antibody host) with 1% BSA to minimize non-specific binding. Third, incubation conditions significantly impact specificity—overnight incubation at 4°C typically provides better signal-to-noise ratio than shorter incubations at room temperature. Fourth, washing steps should be thorough (at least 3-5 washes of 5 minutes each) using PBS with 0.05-0.1% Tween-20 to remove unbound antibody. Fifth, mounting media selection is critical for FITC preservation—anti-fade reagents containing DABCO or proprietary anti-fade compounds help maintain signal intensity during imaging and storage. Sixth, autofluorescence reduction techniques should be employed, particularly when working with tissues with high intrinsic fluorescence (like lung or kidney), using treatments such as Sudan Black B (0.1-0.3%) or commercial autofluorescence quenchers. Finally, image acquisition parameters should be optimized to maximize specific signal while minimizing background, using exposure settings determined by proper controls.

Experimental Design Questions

• What are the appropriate controls needed when using FITC-conjugated MYL9 antibodies?

  A comprehensive control strategy for experiments using FITC-conjugated MYL9 antibodies should include multiple elements to ensure data validity. Primary negative controls must include: (1) Isotype controls using FITC-conjugated non-specific IgG from the same species (rabbit) at matching concentrations to assess non-specific binding; (2) Secondary-only controls (if using indirect detection methods) to evaluate background from secondary reagents; and (3) Unstained samples to establish baseline autofluorescence. Positive controls should include: (1) Tissues or cells known to express MYL9 at detectable levels, such as smooth muscle cells; (2) For phospho-specific MYL9(Thr19) antibodies, samples treated with phosphatase inhibitors to maximize phosphorylation levels; and (3) Comparison with unconjugated primary antibody detected with secondary antibodies to verify that direct conjugation doesn't alter specificity. Additional validation controls should include: (1) Peptide competition/blocking experiments using the immunizing peptide; (2) Parallel staining with alternative antibody clones targeting different MYL9 epitopes; and (3) For studies examining MYL9's role in disease contexts like NSCLC or Kawasaki disease, appropriate disease and normal tissue controls should be included to confirm previously reported expression patterns .

• How should experiments be designed to study MYL9's role in the EMT pathway?

  Designing experiments to investigate MYL9's role in the epithelial-mesenchymal transition (EMT) pathway requires a multifaceted approach based on recent findings that MYL9 suppresses EMT in non-small-cell lung cancer (NSCLC) . First, expression analysis should compare MYL9 levels across epithelial and mesenchymal states, using both The Cancer Genome Atlas (TCGA) bioinformatics data and experimental validation in cell lines. Second, gain-and-loss-of-function studies are essential—both MYL9 knockdown via siRNA/shRNA and overexpression experiments should be performed to establish causality in EMT regulation. Third, phenotypic assays measuring migration (scratch wound healing assays), invasion (transwell assays), and morphological changes should quantify the functional impact of MYL9 modulation. Fourth, molecular marker analysis using Western blotting and immunofluorescence should assess how MYL9 manipulation affects key EMT markers including E-cadherin, vimentin, N-cadherin, and transcription factors like ZEB1. Fifth, interaction studies using coimmunoprecipitation and GST pull-down assays should verify the binding between MYL9 and MYO19, which has been identified as a critical interaction mediating MYL9's effects on EMT . Sixth, rescue experiments where MYO19 is overexpressed in the presence of MYL9 overexpression can confirm the mechanistic relationship. Finally, pathway analysis using gene set enrichment analysis (GSEA) can identify additional signaling networks connected to MYL9-mediated EMT regulation.

• What sample preparation techniques optimize MYL9 detection in different tissue types?

  Optimizing MYL9 detection across diverse tissue types requires tailored sample preparation strategies that address tissue-specific challenges. For formalin-fixed paraffin-embedded (FFPE) tissues, antigen retrieval is critical—heat-induced epitope retrieval using citrate buffer (pH 6.0) for 20 minutes at 95-98°C typically provides good results for MYL9 detection, with recommended antibody dilutions of 1:50-200 for immunohistochemistry applications . For frozen tissues, brief fixation (10 minutes) with 4% paraformaldehyde helps maintain morphology while preserving epitope accessibility. For lung tissue specifically, where MYL9 has shown significant downregulation in NSCLC , additional steps to reduce autofluorescence are crucial—treatment with 0.1% Sudan Black B in 70% ethanol for 20 minutes can substantially reduce background. In cardiovascular tissues, where MYL9 has been studied in Kawasaki disease , careful attention to vessel morphology preservation during fixation is essential. For cell cultures, paraformaldehyde fixation (4%, 15 minutes) followed by 0.2% Triton X-100 permeabilization (10 minutes) typically yields optimal results, though methanol fixation (-20°C, 10 minutes) may better preserve phospho-epitopes for phospho-MYL9 detection. Timing is particularly critical when studying phosphorylation events—samples should be rapidly fixed to capture the dynamic phosphorylation state of MYL9, and phosphatase inhibitors should be included in all buffers prior to complete fixation.

• How can researchers quantitatively analyze MYL9 expression patterns in IF images?

  Quantitative analysis of MYL9 expression patterns in immunofluorescence images requires robust image acquisition and analytical methodologies. First, standardized image acquisition is essential—microscope settings including exposure time, gain, and offset should be established using controls and maintained constant across all experimental samples. Second, proper sampling strategies should be implemented—for tissue sections, systematic random sampling with at least 5-10 fields per section should be analyzed to account for tissue heterogeneity. Third, appropriate image analysis software (ImageJ/FIJI, CellProfiler, or commercial platforms) should be used to implement segmentation strategies that accurately identify cellular compartments (membrane, cytoplasm, nucleus) for localization analysis. Fourth, quantification parameters must be clearly defined—mean fluorescence intensity (MFI), integrated density, or area of positive staining can be measured depending on the experimental question. Fifth, colocalization analysis using Pearson's or Mander's coefficients should be performed when examining MYL9's interaction with other proteins like MYO19 . Sixth, normalization approaches should be established—using housekeeping proteins or total protein staining (like DAPI for nuclear normalization) to account for variations in cell number and tissue thickness. Finally, statistical analysis should incorporate appropriate tests that account for the distribution of the data, with multiple comparisons correction when examining differences across experimental groups. For phospho-MYL9 analysis, ratio imaging comparing phospho-specific to total MYL9 staining provides the most informative measure of activation state.

Data Interpretation Questions

• How can researchers distinguish between active (phosphorylated) and inactive MYL9 in immunofluorescence images?

  Distinguishing between active (phosphorylated) and inactive forms of MYL9 in immunofluorescence images requires specific technical and analytical approaches. First, phospho-specific antibodies targeting the Thr19 phosphorylation site, such as the MYL9(Thr19) polyclonal antibody , provide direct visualization of the active form. Second, dual immunostaining combining phospho-specific and total MYL9 antibodies (using different fluorophores) enables calculation of phosphorylation ratios within the same cells or tissue regions—this normalized approach controls for variations in total MYL9 expression levels. Third, phosphorylation-associated conformational changes in MYL9 can sometimes be detected through differential accessibility of certain epitopes—comparing staining patterns between antibodies targeting different regions may reveal activation-specific patterns. Fourth, functional correlation with contractile structures provides contextual evidence—colocalization of phospho-MYL9 with stress fibers or contractile rings (visualized with phalloidin staining) supports its active status. Fifth, physiological or pharmacological manipulations can validate phospho-specificity—treatment with phosphatase inhibitors (calyculin A, okadaic acid) should increase phospho-MYL9 signal, while myosin light chain kinase inhibitors (ML-7, ML-9) should decrease it. Finally, quantitative image analysis should include intensity-based measurements within defined cellular regions and colocalization metrics with known interacting partners like MYO19 . When interpreting data, it's important to recognize that phosphorylation is dynamic and can be lost during sample processing unless appropriate phosphatase inhibitors are included.

• What patterns of MYL9 localization correlate with specific cellular processes?

  MYL9 localization patterns in immunofluorescence images provide valuable insights into its functional roles in various cellular processes. In contractile events, phosphorylated MYL9 typically shows strong colocalization with actin stress fibers, particularly during cell migration and morphological changes. During cytokinesis, a process explicitly linked to MYL9 function , active MYL9 concentrates at the cleavage furrow and contractile ring, appearing as an intense band between separating daughter cells. In epithelial cells undergoing EMT (a process MYL9 has been shown to suppress in NSCLC ), changes in MYL9 distribution often correlate with reorganization of the cytoskeleton—in epithelial states, a more cortical distribution may be observed, while mesenchymal transformation may show redistribution to stress fibers throughout the cytoplasm. In cellular locomotion, another documented function of MYL9 , polarized distribution with enrichment at the cell rear drives retraction forces. During receptor capping events, MYL9 transiently concentrates beneath clustered membrane receptors. In platelet activation, a process relevant to MYL9's role in Kawasaki disease , diffuse cytoplasmic distribution changes to a peripheral pattern associated with shape change. These localization patterns can be quantified using line scan analysis across cellular regions, intensity correlation with markers of specific structures, or advanced approaches like proximity ligation assays to detect specific protein-protein interactions in situ.

• How should alterations in MYL9 expression be interpreted in cancer tissue samples?

  Interpreting alterations in MYL9 expression in cancer tissue samples requires careful consideration of context-specific factors and integration with other molecular markers. In non-small-cell lung cancer (NSCLC), downregulation of MYL9 has been documented both in vivo and in cell cultures , suggesting a tumor-suppressive role. When analyzing such samples, quantification should account for heterogeneity within the tumor—comparing expression across tumor regions, invasive fronts, and adjacent normal tissue can reveal important patterns. The relationship between MYL9 downregulation and EMT marker expression (E-cadherin loss, vimentin increase) should be assessed, as research indicates MYL9 suppresses EMT in NSCLC . Correlation with MYO19 expression is particularly informative given their antagonistic relationship—higher MYO19/lower MYL9 expression correlates with worse prognosis in NSCLC patients . Beyond expression levels, phosphorylation status of remaining MYL9 provides insights into its functional activity. For comprehensive interpretation, MYL9 data should be integrated with clinicopathological parameters including tumor stage, differentiation status, and patient outcomes. In contrast to NSCLC, other cancer types may show different patterns—in gastric cancer, for example, MYL9 has been reported to function as a prognostic biomarker associated with EMT promotion , highlighting the importance of cancer-type specificity in interpretation. Multivariate analysis incorporating MYL9 with established prognostic markers can determine its independent contribution to patient outcomes.

• How can MYL9 expression data be integrated with other biomarkers in inflammatory conditions?

  Integrating MYL9 expression data with other biomarkers in inflammatory conditions requires methodological approaches that align with MYL9's emerging role as an inflammatory mediator. In Kawasaki disease (KD), where plasma MYL9 levels are significantly elevated during acute phases , correlation analysis with established inflammatory markers provides context—positive correlations with white blood cell (WBC) counts and C-reactive protein (CRP) levels have been documented, along with negative correlations with platelet counts . Multiparameter analysis should categorize patients based on treatment response patterns—MYL9 levels decrease following intravenous immunoglobulin (IVIG) therapy in responders but remain elevated in non-responders, suggesting potential as a predictive biomarker . Longitudinal sampling is crucial for monitoring dynamic changes—MYL9 levels measured before treatment, immediately after, and during follow-up provide trajectory information more valuable than single time points. Multiplex immunoassays combining MYL9 with other inflammatory cytokines and platelet activation markers can establish broader inflammatory signatures. In tissue analyses, dual immunofluorescence examining MYL9 colocalization with immune cell markers (CD45, CD3, CD68) helps identify cellular sources and targets of MYL9 in inflammatory contexts. For mechanistic understanding, correlating MYL9 expression with CD69-expressing inflammatory leukocytes may be particularly informative given the proposed "CD69-Myl9 system" where inflammatory leukocytes utilize 'Myl9 nets' to migrate into and remain within inflamed tissues . Additionally, ratio analyses comparing MYL9 with anti-inflammatory markers may provide insights into inflammatory balance and resolution potential.