MYO1E Antibody

What is MYO1E and what cellular functions does it serve?

MYO1E, also known as Myosin-Ie, belongs to the nonmuscle class I myosins, a subgroup of the unconventional myosin protein family. It functions as an actin-based molecular motor with ATPase activity . This cytoplasmic protein participates in intracellular movement and membrane trafficking, with specific roles in clathrin-mediated endocytosis and the movement of clathrin-coated vesicles . MYO1E is essential for normal morphology of the glomerular basement membrane and the development of foot processes by kidney podocytes . In dendritic cells, MYO1E may control the movement of class II-containing cytoplasmic vesicles along the actin cytoskeleton by connecting them with the actin network via ARL14EP and ARL14 .

Where is MYO1E expressed and how does its expression pattern inform antibody selection?

MYO1E is expressed in various tissues and cell lines. According to the search results, MYO1E protein has been detected in:

MYO1E is highly expressed in various malignancies, including pancreatic adenocarcinoma (PAAD), lung adenocarcinoma (LUAD), glioblastoma (GBM), colon adenocarcinoma (COAD), breast invasive carcinoma (BRCA), and head and neck squamous carcinoma (HNSC) . When selecting antibodies, consider the expression levels in your target tissue and the specific subcellular localization pattern expected. In podocytes, MYO1E localizes to punctate structures, some of which are labeled with anti-synaptopodin antibodies, and is enriched in areas of cell-cell contacts .

What are the available types of MYO1E antibodies and their characteristics?

Based on the search results, there are both polyclonal and monoclonal antibodies available for MYO1E research:

Both types have advantages: polyclonal antibodies often provide higher sensitivity by recognizing multiple epitopes, while monoclonal antibodies offer higher specificity and consistency between batches . Selection should be based on your specific experimental requirements and the nature of your samples.

What are the recommended dilutions and applications for MYO1E antibodies?

The optimal dilutions depend on both the specific antibody and the application. Based on the search results, here are the recommended dilutions for common applications:

Always titrate the antibody in your specific testing system to determine the optimal concentration for your experimental conditions . The wide range of recommended dilutions reflects the variability in antibody performance across different sample types and protocols.

How can I validate the specificity of MYO1E antibodies in my experiments?

To ensure the specificity of MYO1E antibodies, implement the following validation strategies:

• Positive controls: Use cell lines known to express MYO1E such as HeLa, Jurkat, or HEK-293 cells as positive controls .

• Knockout/knockdown validation: Use MYO1E knockout mice tissues or cells with siRNA knockdown of MYO1E as negative controls. Studies have successfully used this approach to validate antibody specificity .

• Western blot molecular weight verification: Confirm that the detected band appears at the expected molecular weight (approximately 127 kDa, though observed weight may range from 120-150 kDa) .

• Immunofluorescence pattern verification: Compare your staining pattern with published patterns. For example, in podocytes, MYO1E localizes to punctate structures and cell-cell contacts .

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide to block specific binding sites before application to your sample.

What sample preparation techniques maximize MYO1E detection in different assays?

For optimal MYO1E detection across different assay types:

Western Blot:

• Extract total proteins using RIPA buffer

• Add 5× loading buffer and heat at 95°C for 10 minutes

• Separate proteins using 10% SDS-PAGE

• Use 0.45 μm PVDF membranes for transfer

• Block with 5% skim milk at room temperature for 2 hours

Immunohistochemistry:

• For MYO1E detection in kidney tissue, use antigen retrieval with TE buffer pH 9.0

• Alternatively, antigen retrieval may be performed with citrate buffer pH 6.0

• For podocyte visualization, co-staining with synaptopodin can help identify MYO1E-positive structures

Immunofluorescence:

• MYO1E is primarily cytoplasmic, sometimes enriched in cell-cell contacts

• Co-labeling with β-catenin can help identify MYO1E at cell-cell junctions

• Include appropriate controls (e.g., nonimmune IgG) to confirm staining specificity

How does MYO1E function in kidney podocytes and what methodologies best elucidate this role?

MYO1E plays a critical role in podocyte function and normal glomerular filtration. MYO1E-knockout mice exhibit proteinuria, chronic renal injury, and kidney inflammation . At the ultrastructural level, disruption of MYO1E leads to thickened and disorganized glomerular basement membrane and flattened podocyte foot processes .

To investigate MYO1E's role in podocytes, researchers have employed several methodologies:

• Genetic manipulation: Generate Myo1e-knockout mice to study systemic effects of MYO1E deficiency .

• Zebrafish models: Specific knockdown of MYO1E in zebrafish results in pericardial edema and pronephric cysts, demonstrating conservation of MYO1E function across species .

• Immortalized podocyte cell lines: Conditional immortalization allows for in vitro study of MYO1E's effects on podocyte morphology, actin cytoskeleton organization, and cellular functions .

• Functional assays: Measure cell proliferation, migration, endocytosis, and adhesion to assess how MYO1E affects podocyte functions .

• Ultrastructural analysis: Electron microscopy reveals changes in glomerular basement membrane and podocyte foot processes in MYO1E-deficient models .

What role does MYO1E play in immune cell function and how can this be studied with MYO1E antibodies?

MYO1E regulates specific aspects of macrophage and dendritic cell (DC) function in response to Toll-like receptor 4 (TLR4) stimulation. MYO1E-deficient macrophages and DCs show impairments in:

• Cell spreading: Myo1e-deficient macrophages exhibit reduced spreading in response to LPS after 2h and 8h .

• MHC-II surface expression: 24 hours after LPS stimulation, MYO1E-deficient DCs show a significant reduction (approximately 20%) in MHC-II surface protein compared to wild-type cells .

• T-cell activation: The capacity of antigen-presenting cells lacking MYO1E to stimulate antigen-specific CD4+ T-cell proliferation is impaired .

To study these functions, researchers can:

• Use flow cytometry to measure surface expression of MHC-II and co-stimulatory molecules on LPS-stimulated wild-type vs. MYO1E-deficient cells

• Employ co-culture systems with antigen-specific T cells to assess the antigen presentation capacity of MYO1E-deficient APCs

• Use live cell imaging with fluorescently tagged MYO1E to track its dynamics during immune cell activation

• Implement biochemical approaches to identify MYO1E-interacting proteins in immune cells

How is MYO1E implicated in cancer research and what experimental approaches reveal its mechanistic contributions?

MYO1E is overexpressed in multiple cancer types and may serve as a potential biomarker, particularly in pancreatic adenocarcinoma (PAAD). Recent research has revealed:

• Elevated expression: MYO1E mRNA and protein expression levels are higher in PAAD tissues than in normal tissues .

• Prognostic significance: High MYO1E expression is associated with poor prognosis in PAAD patients, correlating with pathological stage .

• Functional impact: In vitro suppression of MYO1E expression inhibits pancreatic adenocarcinoma cell proliferation, invasion, and migration .

• Pathway involvement: MYO1E is linked to multiple tumor-related mechanisms in PAAD, including the PI3K-AKT signaling pathway and ECM-receptor interaction .

• Immune microenvironment: MYO1E expression positively correlates with tumor immune cell infiltration and is associated with tumor chemokines/receptors and immune checkpoints .

Experimental approaches to study MYO1E in cancer include:

• Immunohistochemistry to compare MYO1E expression between tumor and normal tissues

• siRNA knockdown of MYO1E followed by functional assays (proliferation, migration, invasion)

• RNA-seq analysis to identify genes differentially expressed upon MYO1E modulation

• Co-immunoprecipitation to identify MYO1E-interacting proteins in cancer cells

• Tumor xenograft models to study the effects of MYO1E modulation on tumor growth in vivo

What are common technical challenges when using MYO1E antibodies and how can they be addressed?

When working with MYO1E antibodies, researchers may encounter several technical challenges:

• High background in immunostaining:

  • Solution: Optimize blocking conditions (try 5% BSA or 5% normal serum from the same species as the secondary antibody)

  • Increase washing steps and duration

  • Titrate primary antibody concentration

• Multiple bands in Western blot:

  • Solution: Optimize lysis conditions to prevent protein degradation

  • Include protease inhibitors in extraction buffers

  • Verify that observed bands match the expected molecular weight (127 kDa)

  • Consider that the observed molecular weight may range from 120-150 kDa

• Weak or no signal in Western blot:

  • Solution: Ensure adequate protein loading (10-30 μg total protein)

  • Optimize transfer conditions for high molecular weight proteins

  • Try different antigen retrieval methods for IHC (TE buffer pH 9.0 or citrate buffer pH 6.0)

  • Consider the expression level of MYO1E in your sample

• Cross-reactivity with other myosin family members:

  • Solution: Use knockout/knockdown controls

  • Compare staining patterns with other myosin antibodies (e.g., nonmuscle myosin IIA shows different localization)

  • Validate with multiple antibodies targeting different epitopes of MYO1E

How can I differentiate between MYO1E and other closely related myosin family members?

Distinguishing MYO1E from other myosin family members requires careful experimental design:

• Epitope selection: Choose antibodies targeting unique regions of MYO1E. Note that MYO1E (formerly called MYO1C) is distinct from the MYO1C gene located on chromosome 17 .

• Localization patterns: Different myosins show distinct subcellular localization patterns. For example, MYO1E localizes to punctate structures and cell-cell contacts, while nonmuscle myosin IIA (MYO2A) localizes along actin stress fibers .

• Molecular weight verification: MYO1E has a calculated molecular weight of 127 kDa, which may help distinguish it from other myosin family members on Western blots .

• Genetic approaches: Use siRNA or CRISPR targeting MYO1E specifically to confirm antibody specificity. Researchers have successfully used siRNA knockdown of MYO1E with sequences such as:

  • si-MYO1E#1: sense 5′-CAGAAGCAACUACCUCUGAAA-3′; antisense 5′-UUUCAGAGGUAGUUGCUUCUG-3′

  • si-MYO1E#2: sense 5′-CCUCAUAGAGAACAAAGUGAA-3′; antisense 5′-UUCACUUUGUUCUCUAUGAGG-3′

• Co-localization studies: Use known interacting partners or structures specific to MYO1E (e.g., synaptopodin in podocytes) to confirm the identity of the detected protein .

What considerations should be made when designing experiments to study MYO1E in different disease models?

When designing experiments to study MYO1E in disease models, consider:

• Model selection:

  • For kidney disorders: MYO1E knockout mice show proteinuria and glomerular abnormalities, mimicking human focal segmental glomerulosclerosis

  • For immune function: MYO1E-deficient mice can be used to study macrophage and dendritic cell functions

  • For cancer: Patient-derived xenografts or cell lines with manipulated MYO1E expression can model its role in tumor progression

  • For developmental studies: Zebrafish models with MYO1E knockdown show pericardial edema and pronephric cysts

• Endpoint measurements:

  • For kidney disease: Measure proteinuria, kidney/body weight ratio, ultrastructural changes in glomeruli

  • For immune function: Assess cell spreading, MHC-II surface expression, T-cell activation capacity

  • For cancer: Evaluate cell proliferation, invasion, migration, and gene expression changes

• Control selection:

  • Include age-matched wild-type controls for animal studies

  • Use appropriate vector controls for gene manipulation studies

  • Include isotype controls for antibody experiments

• Mechanistic investigations:

  • Consider the role of MYO1E in actin cytoskeleton organization

  • Evaluate effects on membrane trafficking and endocytosis

  • Assess interactions with known binding partners

• Translational relevance:

  • Correlate findings in model systems with human disease samples

  • Consider how MYO1E expression or function might be therapeutically targeted

By carefully addressing these considerations, researchers can design robust experiments to elucidate MYO1E's role in various disease processes.