myo1c Antibody

What is MYO1C and what cellular functions does it participate in?

MYO1C (myosin IC) is an unconventional myosin protein with a molecular weight of approximately 121.7 kDa and 1063 amino acid residues in humans. It functions as a molecular motor that converts chemical energy into mechanical work and is involved in multiple cellular processes, including:

• Chromatin remodeling and regulation of cell migration

• Intracellular transport and membrane trafficking

• Linking the actin cytoskeleton to cellular membranes through phosphoinositide binding

• Glucose transporter recycling in response to insulin

• Transcriptional regulation (particularly isoform 3)

MYO1C is abundantly expressed in murine B lymphocytes, where it localizes primarily to the plasma membrane, especially in peripheral processes such as microvilli. It concentrates at growing membrane protrusions during B cell spreading and is actively recruited to the immune synapse .

What is the subcellular localization of MYO1C?

MYO1C exhibits distinct subcellular localization patterns that reflect its diverse functions:

• Nucleus: Involved in transcriptional regulation and ribosomal gene expression

• Cytoplasmic vesicles: Participates in vesicular transport

• Cytoplasm: General distribution throughout the cytoplasmic compartment

• Plasma membrane: Particularly enriched in membrane protrusions and microvilli

In B lymphocytes, MYO1C shows strong colocalization with MHC-II molecules, particularly after cross-linking of these molecules, suggesting a role in membrane protein anchoring or sorting .

What experimental applications are MYO1C antibodies commonly used for?

MYO1C antibodies are utilized in multiple experimental applications:

These applications are supported by multiple vendors offering over 110 different MYO1C antibodies with various specificities and applications .

How should I select the appropriate MYO1C antibody for my experiment?

When selecting a MYO1C antibody, consider the following factors:

• Experimental application (WB, IHC, ICC, IP, ELISA)

• Species reactivity (human, mouse, rat)

• Antibody type (monoclonal vs. polyclonal)

• Clonal information (e.g., EPR14771)

• Epitope recognition region (N-terminal, C-terminal, or specific domain)

• Validation data for your specific application

• Published literature using the antibody

For detecting direct interactions between MYO1C and other proteins (like rhodopsin), select antibodies validated for immunoprecipitation and confirmed not to interfere with protein binding domains .

How can I validate MYO1C antibody specificity in my experimental system?

Validating MYO1C antibody specificity requires multiple complementary approaches:

• Genetic controls: Use tissues or cells from Myo1c-KO models as negative controls. Complete loss of signal in western blotting and immunostaining of tissues from Myo1c-KO mice confirms antibody specificity .

• siRNA knockdown validation: Transfect cells with siRNAs targeting Myo1c (e.g., SMARTpools from Dharmacon, catalog no. L-015121-00-0005) and confirm reduced signal in western blotting compared to controls transfected with non-targeting siRNA (e.g., Firefly luciferase targeted siRNA) .

• Multiple antibodies approach: Use at least two different antibodies recognizing distinct epitopes of MYO1C and confirm similar staining patterns.

• Peptide competition: Pre-incubate the antibody with the immunizing peptide to block specific binding and observe signal elimination .

What protocols are recommended for studying MYO1C-protein interactions?

To investigate MYO1C interactions with other proteins, these approaches have been successful:

• Co-immunoprecipitation (Co-IP):

  • Use MYO1C antibodies to pull down protein complexes from tissue/cell lysates

  • Perform reciprocal IP with antibodies against suspected interacting proteins

  • Western blot precipitated complexes to confirm interaction

• Direct binding overlay assay:

  • Immunoprecipitate potential interacting proteins (e.g., rhodopsin)

  • Separate by SDS-PAGE and transfer to membrane

  • Probe membrane with purified recombinant MYO1C protein (full-length or domain-specific)

  • Detect bound MYO1C with MYO1C antibody to confirm direct binding

For example, researchers demonstrated direct interaction between MYO1C and rhodopsin using both co-IP from retinal lysates and a direct binding overlay assay with purified recombinant MYO1C protein .

How can I study the role of MYO1C in cellular processes using functional inhibition approaches?

Several complementary approaches can be used to study MYO1C function:

• siRNA-mediated knockdown:

  • Transfect cells with Myo1c-targeted siRNAs

  • Verify knockdown efficiency by western blot

  • Assess phenotypic changes in cellular processes (e.g., reduced VWF secretion following Myo1c knockdown)

• Dominant-negative constructs:

  • Express mutant forms of MYO1C that interfere with endogenous protein function

  • The G108R mutation creates a rigor mutant by changing a conserved residue in the nucleotide binding region

  • This approach revealed alterations in B cell spreading and antigen-presenting abilities

• Small molecule inhibitors:

  • Use pentachloropseudilin (PCLP) to inhibit class I myosins

  • Treat cells with 5-20 μM PCLP for 30 minutes to 16 hours before functional assays

  • This approach revealed reduced VWF secretion in endothelial cells

How do I detect different MYO1C isoforms in my samples?

MYO1C has three reported isoforms generated through alternative splicing. To distinguish between them:

• Isoform-specific antibodies:

  • Select antibodies raised against regions unique to specific isoforms

  • Verify isoform specificity using recombinant proteins or cells expressing single isoforms

• Western blotting with high-resolution gels:

  • Use 6-8% SDS-PAGE gels for optimal separation of high molecular weight isoforms

  • Extended running times may be necessary to resolve small size differences

  • Confirm bands using isoform-specific antibodies if available

• RT-PCR for isoform-specific mRNA detection:

  • Design primers spanning unique exon junctions for each isoform

  • Quantify relative expression levels of different isoforms

What methods can be used to study MYO1C's interaction with membrane phospholipids?

MYO1C interacts with membrane phospholipids, particularly phosphatidylinositol 4,5-bisphosphate (PIP2), through its pleckstrin homology domain. To study these interactions:

• Lipid overlay/binding assays:

  • Spot purified lipids on membranes

  • Incubate with recombinant MYO1C protein

  • Detect bound MYO1C with antibodies

• Liposome co-sedimentation assays:

  • Generate liposomes containing specific phospholipids

  • Incubate with purified MYO1C

  • Pellet liposomes and analyze bound proteins by western blotting

• Pleckstrin homology domain mutation analysis:

  • Express wild-type or mutant MYO1C (with mutations in the PH domain)

  • Compare recruitment to membranes or lipid-rich structures

  • This approach revealed that MYO1C is recruited to fused secretory organelles via its PH domain in an actin-independent process

How can I assess MYO1C's role in cellular function using live-cell imaging approaches?

To visualize MYO1C dynamics in living cells:

• GFP-tagged MYO1C constructs:

  • Express full-length MYO1C-GFP or domain-specific constructs (e.g., GFP-MYO1C-790-1028 for the tail/cargo domain)

  • Use spinning disk confocal or TIRF microscopy for high-resolution imaging

  • Track localization during cellular processes like membrane protrusion or vesicle trafficking

• Photoactivatable or photoconvertible fusion proteins:

  • Create MYO1C fusions with tags like PA-GFP or mEos

  • Activate specific pools of the protein and track movement over time

  • Quantify dynamics using fluorescence recovery after photobleaching (FRAP)

• Dual-color imaging with cellular markers:

  • Co-express MYO1C-GFP with markers for cellular compartments

  • Use live-cell compatible antibodies against extracellular epitopes of interacting proteins

  • For example, this approach could visualize MYO1C recruitment to exocytic sites during von Willebrand factor secretion

What are emerging areas of MYO1C research where antibodies will be valuable tools?

Several promising research directions for MYO1C antibodies include:

• Role in immune cell function: Beyond B cells, investigating MYO1C's role in other immune cell types and immunological processes, particularly given its involvement in cytoskeleton rearrangements and membrane protein organization .

• Secretory pathways: Further exploring MYO1C's role in regulated secretion across different cell types, building on findings in endothelial cells where it augments von Willebrand factor secretion .

• Nuclear functions: Investigating MYO1C's involvement in transcriptional regulation and chromatin remodeling, especially for isoform 3 which associates with transcriptionally active ribosomal genes .

• Therapeutic targeting: Developing tools to modulate MYO1C function in pathological conditions related to secretion, membrane trafficking, or cytoskeletal dynamics.