MYL9 Antibody

What is MYL9 and what is its significance in cellular function?

MYL9 is a myosin regulatory light chain with a canonical length of 172 amino acid residues and a molecular weight of approximately 19.8 kDa. It functions as a regulatory subunit that plays a crucial role in both smooth muscle and nonmuscle cell contractile activity through its phosphorylation state. MYL9 is primarily localized in the cytoplasm and exists in two alternatively spliced isoforms. It's a critical component in regulating cellular contraction, motility, and structural integrity. In normal physiology, MYL9 is particularly important for smooth muscle function, with MYL9 deficiency in mice causing lethal abnormalities in multiple organs including the bladder, small intestine, and lungs .

How do I select the appropriate MYL9 antibody for my research?

Selection of an appropriate MYL9 antibody requires careful consideration of several factors:

• Specificity concerns: MYL9 shares >96% amino acid homology with MYL12A and MYL12B, leading to significant cross-reactivity of antibodies. Consider using genetic knockdown controls to validate specificity.

• Application compatibility: Verify that the antibody has been validated for your specific application (Western blot, IHC, IF, ELISA).

• Host species: Select an antibody raised in a species that complements your experimental system to avoid cross-reactivity in multi-antibody studies.

• Epitope location: For phosphorylation studies, ensure the antibody recognizes the appropriate phosphorylated or non-phosphorylated form.

• Tissue expression: MYL9 shows tissue-specific expression patterns. For instance, it's restricted to the muscularis propria of the small intestine and bladder, and in smooth muscle layers of bronchi and major vessels .

What are the common applications for MYL9 antibodies in research?

MYL9 antibodies are utilized across multiple research applications:

• Western blotting: The most widely used application for detecting MYL9 protein expression levels in cell and tissue lysates.

• Immunohistochemistry (IHC): Used to visualize MYL9 expression patterns in tissue sections, particularly valuable for studying smooth muscle and cancer tissue samples.

• Immunofluorescence (IF): Enables subcellular localization studies of MYL9 protein.

• ELISA: Quantitative measurement of MYL9 in biological samples.

• Chromatin immunoprecipitation (ChIP): When studying transcriptional regulation involving MYL9.

Each application requires specific optimization parameters including antibody concentration, incubation time, and buffer conditions .

How can I effectively validate MYL9 antibody specificity?

Validating MYL9 antibody specificity is particularly challenging due to high homology with related proteins. A comprehensive validation approach includes:

• Genetic knockdown validation: Using siRNA targeting MYL9 (as demonstrated in research where siRNA-MYL9-1 and siRNA-MYL9-2 were employed to silence MYL9 expression) provides the most conclusive validation method .

• Overexpression control: Testing antibody reactivity in systems with ectopic MYL9 overexpression (e.g., Ov-MYL9 transfected cells) .

• Knockout tissues: If available, tissues from MYL9 knockout models serve as gold-standard negative controls. Studies using MYL9-deficient mice have shown the complete absence of antibody signal in tissues that normally express MYL9 .

• Multiple antibody validation: Using different antibodies targeting distinct epitopes of MYL9.

• Western blot molecular weight verification: Confirming reactivity at the expected 19.8 kDa range.

• Cross-reactivity assessment: Evaluating potential cross-reactivity with MYL12A and MYL12B by comparing expression patterns with known tissue-specific distribution patterns .

What are the optimal conditions for Western blotting with MYL9 antibodies?

Successful Western blotting for MYL9 requires careful optimization:

• Protein extraction: Use RIPA lysis buffer containing phenylmethanesulfonyl fluoride and phosphatase inhibitors (at a ratio of 100:1:1) to effectively extract total protein while preserving phosphorylation states.

• Protein loading: 30 μg of protein is typically sufficient for detection in cells with moderate MYL9 expression.

• Gel percentage: 10% SDS-PAGE gels provide optimal separation for the 19.8 kDa MYL9 protein.

• Transfer conditions: Standard PVDF membranes are suitable for MYL9 transfer.

• Blocking: 5% bovine serum albumin in TBST for 1 hour at 37°C minimizes background.

• Primary antibody: Incubate with anti-MYL9 antibody (1:1000 dilution is typically effective) overnight at 4°C.

• Secondary antibody: HRP-conjugated anti-mouse or anti-rabbit IgG (depending on primary antibody host) at 37°C for 1 hour.

• Detection: Standard ECL detection systems are sufficient for visualizing MYL9 bands.

Include appropriate loading controls such as β-tubulin or GAPDH for normalization .

What approaches are recommended for analyzing MYL9 gene expression?

For comprehensive MYL9 gene expression analysis:

• RT-qPCR protocol:

  • Extract total RNA using TRIzol reagent

  • Synthesize cDNA from 1 μg of total RNA using reverse transcriptase

  • Perform qPCR using SYBR Green with specific primers

  • Recommended primer sequences:

    • MYL9 forward: 5′-GCCACATCCAATGTCTTCGC-3′

    • MYL9 reverse: 5′-GCGTTGCGAATCACATCCTC-3′

  • Use GAPDH as internal control

  • Calculate relative expression using the 2^-ΔΔCt method

• Thermocycling conditions:

  • Initial denaturation at 95°C for 10 minutes

  • 45 cycles of: 95°C for 15s, 60°C for 15s, and 72°C for 10s

  • Confirm specificity with melting curve analysis

• Expression normalization: For comparative studies, set the expression in control samples as 1 (such as peritumoral tissues in cancer studies) .

How is MYL9 involved in colorectal cancer progression?

MYL9 has been identified as a key regulator in colorectal cancer (CRC) progression through multiple mechanisms:

• Overexpression pattern: MYL9 protein and mRNA expression is significantly upregulated in colorectal cancer cell lines (SW480, SW620, HT-29, and HCT116) compared to normal colorectal epithelial cells (NCM460) .

• Functional impacts on cancer hallmarks:

  • Proliferation: MYL9 knockdown using siRNA significantly decreases CRC cell proliferation, while overexpression enhances it.

  • Migration and invasion: MYL9 silencing inhibits, while overexpression promotes, the migration and invasion capabilities of CRC cells.

  • Metastasis-related proteins: MYL9 modulates the expression of matrix metalloproteinases (MMP2 and MMP9), with knockdown decreasing and overexpression increasing these metastasis-promoting proteins .

• Molecular mechanism: MYL9 promotes CRC progression by binding to YAP1 and thereby activating Hippo signaling, a pathway critical for tumor growth and metastasis .

• Microenvironment regulation: Interestingly, MYL9 is predominantly expressed in cancer-associated fibroblasts (CAFs) rather than CRC cells themselves. In this context, MYL9 regulates the secretion of CCL2 and TGF-β1 from CAFs, which affects the immune microenvironment and CRC progression .

What methodologies are optimal for studying MYL9's role in cancer cell migration and invasion?

To effectively investigate MYL9's impact on cancer cell migration and invasion:

• Genetic manipulation approaches:

  • siRNA knockdown: Use siRNA-MYL9 constructs (multiple sequences for validation) transfected into cancer cell lines.

  • Overexpression systems: Employ Ov-MYL9 plasmids with appropriate empty vector controls .

• Migration assays:

  • Transwell migration assay: Seed cells in serum-free medium in the upper chamber and measure migration toward chemoattractant in the lower chamber after 24-48 hours.

  • Wound healing assay: Create a scratch in a confluent monolayer and monitor closure over time .

• Invasion assays:

  • Boyden chamber assay: Similar to Transwell but with Matrigel coating to mimic extracellular matrix.

  • 3D spheroid invasion assay: Grow cancer cells as spheroids embedded in matrix to assess invasion in a more physiologically relevant context .

• Co-culture systems for stromal interaction studies:

  • Indirect co-culture: Collect conditioned medium (CM) from MYL9-manipulated CAFs and apply to cancer cells.

  • Direct co-culture: Use transwell systems (0.4 μm pore size) to allow paracrine interactions without cell mixing .

• Colony formation assay:

  • Seed 800 cells/well in 6-well plates with conditioned medium from MYL9-manipulated cells.

  • Culture for 10-14 days, then fix, stain, and quantify colony formation .

How does MYL9 influence cancer metabolism, particularly glycolysis?

MYL9 has been discovered to significantly impact cancer cell metabolism, particularly aerobic glycolysis (the Warburg effect):

• Glycolytic enhancement: Research has demonstrated that MYL9 promotes squamous cervical cancer (SCC) migration and invasion specifically by enhancing aerobic glycolysis .

• Molecular mechanism: MYL9 increases the activity of the JAK2/STAT3 signaling pathway, which is known to regulate metabolic reprogramming in cancer cells .

• Glycolytic markers regulated by MYL9:

  • GLUT1 (glucose transporter 1): Facilitates increased glucose uptake

  • HK2 (hexokinase 2): Catalyzes the first rate-limiting step of glycolysis

  • LDHA (lactate dehydrogenase A): Converts pyruvate to lactate, enabling continued glycolytic flux

• Measurement approaches:

  • Lactate production assays to quantify glycolytic output

  • Glucose consumption assays

  • Expression analysis of glycolytic enzymes via Western blot and RT-qPCR

  • Metabolic flux analysis using isotope-labeled glucose

• Therapeutic implications: The connection between MYL9 and glycolysis suggests that targeting MYL9 could potentially disrupt cancer metabolism, offering a novel therapeutic strategy.

How can researchers address the challenge of MYL9 antibody cross-reactivity with MYL12A and MYL12B?

Addressing MYL9 antibody cross-reactivity requires sophisticated experimental design:

• Tissue selection strategy: Leverage differential expression patterns. For instance, bladder and intestine tissues express high levels of MYL9 but low levels of MYL12A and MYL12B, making them suitable for MYL9-focused studies .

• Reporter gene systems: Consider using genetic models with reporter genes like the LacZ-knockin/knockout mouse model, which allows precise mapping of MYL9 expression without relying solely on antibody detection .

• RNA-based validation: Complement protein studies with RNA analysis techniques (RT-qPCR or RNA-seq) using MYL9-specific primers to verify expression patterns .

• Bioinformatic verification: Compare your findings with tissue expression databases (such as GTEx Portal) to confirm expected distribution patterns for MYL9 versus MYL12A/B .

• Genetic knockout controls: The most definitive approach is using tissue from MYL9 knockout models as negative controls, as demonstrated in studies where antibody staining was detected in wild-type tissues but absent in MYL9-deficient tissues .

What are the implications of studying MYL9 in the tumor microenvironment versus cancer cells?

Research has revealed distinct roles for MYL9 depending on its cellular context:

• Cell-specific expression patterns:

  • In colorectal cancer, MYL9 is predominantly expressed in cancer-associated fibroblasts (CAFs) rather than cancer cells themselves .

  • This spatial distribution creates different functional outcomes than when MYL9 is expressed directly within cancer cells.

• Indirect versus direct effects:

  • CAF-expressed MYL9 indirectly influences tumor biology by regulating secretory profiles (CCL2 and TGF-β1) that then affect cancer cells .

  • Cancer cell-expressed MYL9 directly enhances proliferation, migration, and invasion capabilities .

• Methodological considerations:

  • For tumor microenvironment studies: Co-culture systems, conditioned media experiments, and in vivo models are essential.

  • For direct cellular function: Standard cell-autonomous assays (proliferation, migration) with genetic manipulation are appropriate.

• Therapeutic targeting implications:

  • Stromal MYL9 targeting might disrupt tumor-supporting networks.

  • Cancer cell MYL9 targeting could directly inhibit malignant behaviors.

  • Combination approaches may provide synergistic benefits by addressing both components .

How can researchers integrate MYL9 studies with current immunotherapy research?

Emerging research suggests important connections between MYL9 and anti-tumor immunity:

• Immunosuppressive microenvironment: High MYL9 expression in CAFs is associated with M2 macrophage infiltration, creating an immunosuppressive microenvironment in colorectal cancer that reduces sensitivity to immunotherapy .

• Integration methodologies:

  • Analyze MYL9 expression in relation to immune cell infiltration markers

  • Assess correlation between MYL9 levels and response to immunotherapy in patient cohorts

  • Design combination studies targeting both MYL9 and immune checkpoints

• Experimental approaches:

  • Flow cytometry to characterize immune infiltrates in MYL9-high versus MYL9-low tumors

  • Single-cell RNA sequencing to map MYL9 expression across tumor and immune cell populations

  • Multiplex immunohistochemistry to visualize spatial relationships between MYL9-expressing cells and immune cells

• Mechanistic investigations: Explore how MYL9-regulated factors (like CCL2 and TGF-β1) modulate T cell function, immune checkpoint expression, and antigen presentation .

What strategies can address weak or absent MYL9 antibody signal in Western blots?

When confronting weak or absent MYL9 signals in Western blots:

• Sample preparation optimization:

  • Ensure complete protein extraction using RIPA buffer with protease inhibitors

  • Avoid repeated freeze-thaw cycles of protein samples

  • Add phosphatase inhibitors if studying phosphorylated MYL9

• Antibody condition assessment:

  • Verify antibody storage conditions and expiration date

  • Titrate antibody concentration (try ranges from 1:500 to 1:2000)

  • Extend primary antibody incubation (overnight at 4°C is optimal)

• Expression verification:

  • Validate MYL9 expression at mRNA level using RT-qPCR

  • Include positive control samples from tissues known to express MYL9 (bladder, small intestine)

  • Consider the cell types being studied, as MYL9 expression varies significantly across tissues

• Technical optimization:

  • Increase protein loading (up to 50 μg)

  • Use more sensitive detection systems (enhanced chemiluminescence plus)

  • Optimize transfer conditions (lower voltage for longer time for small proteins)

How should conflicting MYL9 expression data between antibody-based and RNA-based methods be reconciled?

When facing discrepancies between protein and RNA detection methods:

• Validation approaches:

  • Perform experiments with multiple antibodies targeting different MYL9 epitopes

  • Use genetic manipulation (siRNA, CRISPR) to confirm specificity

  • Employ alternative protein detection methods (mass spectrometry)

• Biological explanations to consider:

  • Post-transcriptional regulation: MYL9 mRNA can be actively degraded by mechanisms like DROSHA-mediated decay, resulting in mRNA expression without corresponding protein

  • Protein stability differences: Variations in protein half-life can cause disconnects between mRNA and protein levels

  • Cell-type heterogeneity: In tissues with mixed cell populations, bulk RNA methods may detect signal from minority populations

• Resolution strategies:

  • Single-cell approaches to resolve cellular heterogeneity

  • Polysome profiling to assess translation efficiency

  • Protein degradation studies using proteasome inhibitors

• Documentation best practices: Clearly report and discuss discrepancies in publications rather than selectively reporting concordant results .

What are emerging applications for MYL9 antibodies in cancer biomarker development?

MYL9 shows significant potential as a cancer biomarker:

How might researchers approach studying the interplay between MYL9 and epithelial-mesenchymal transition in cancer?

MYL9 has been linked to epithelial-mesenchymal transition (EMT), a critical process in cancer progression:

• Experimental design considerations:

  • Monitor classical EMT markers (E-cadherin, vimentin, Snail, Slug) alongside MYL9 manipulation

  • Use both 2D and 3D culture systems to capture different aspects of EMT

  • Employ live-cell imaging to observe morphological changes in real-time

• Mechanistic investigations:

  • Explore connections between MYL9 and TGF-β signaling, as MYL9 regulates TGF-β1 secretion from CAFs

  • Investigate cytoskeletal remodeling, as MYL9's role in contractile function may directly impact cell shape changes during EMT

  • Study potential interactions with IQGAP1, which binds MYL9 and regulates cytoskeletal dynamics

• In vivo approaches:

  • Develop metastasis models with MYL9 manipulation

  • Analyze circulating tumor cells for MYL9 expression

  • Employ lineage tracing to follow EMT processes in MYL9-altered settings

• Translational relevance:

  • Correlate MYL9 expression with EMT status in patient samples

  • Explore MYL9 as a target for preventing metastasis through EMT inhibition