MTMR14 Antibody

What is MTMR14 and why is it important in biological research?

MTMR14 (myotubularin-related protein 14) is a novel phosphoinositide phosphatase that belongs to the MTM1/MTMRs family, which contains 15 members divided into catalytically active and inactive subgroups . MTMR14 is a catalytically active member that specifically dephosphorylates phosphatidylinositol 3-phosphate (PtdIns3P) and PtdIns(3,5)P2 . The importance of MTMR14 in biological research stems from its diverse roles in multiple cellular processes. It has been demonstrated to maintain normal muscle performance, regulate autophagy, and influence aging in mouse models . Recent studies have also revealed its significant role in metabolism regulation, as MTMR14 knockout mice gain weight earlier than their wild-type counterparts . Additionally, MTMR14 has been identified as a potential inhibitor of vascular smooth muscle cell (VSMC) proliferation, suggesting its importance in preventing neointima formation and restenosis following vessel injury .

What applications are MTMR14 antibodies typically used for in research settings?

MTMR14 antibodies have been validated for several important research applications. The primary applications include:

• Western Blot (WB): MTMR14 antibodies have been extensively tested for WB applications with recommended dilutions typically ranging from 1:500 to 1:4000, depending on the specific antibody and sample type . They have demonstrated positive results in various sample types including human cell lines (Jurkat cells), mouse cell lines (RAW 264.7), and tissue samples from mouse and rat testes .

• Immunohistochemistry (IHC): Some MTMR14 antibodies have been validated for IHC applications with recommended dilutions of 1:50 to 1:200 .

• Immunofluorescence (IF)/Immunocytochemistry (ICC): MTMR14 antibodies have shown positive results in IF/ICC applications, particularly in HeLa cells, with recommended dilutions of 1:50 to 1:500 .

• ELISA: Certain MTMR14 antibodies have been validated for ELISA applications .

• Knockout/Knockdown validation studies: Some MTMR14 antibodies have been used in published studies involving the knockdown or knockout of MTMR14, making them valuable tools for validating gene manipulation experiments .

What is the observed molecular weight of MTMR14 in experimental conditions?

While the calculated molecular weight of MTMR14 is approximately 72 kDa based on its amino acid sequence, the observed molecular weight in experimental conditions typically ranges from 60-72 kDa . This variation may be attributed to post-translational modifications, protein processing, or different isoforms of the protein. When using MTMR14 antibodies for Western blot applications, researchers should expect to observe bands within this range. It's important to note that when working with recombinant MTMR14 proteins that contain fusion tags (such as GST), the observed molecular weight will be higher. For instance, when using a GST-tagged full-length recombinant MTMR14 protein, the GST tag alone contributes approximately 26 kDa to the total molecular weight .

How can MTMR14 antibodies be used to investigate vascular smooth muscle cell proliferation and neointima formation?

MTMR14 antibodies can be instrumental in investigating the role of this protein in vascular smooth muscle cell (VSMC) proliferation and neointima formation through several methodological approaches:

• Expression analysis in vessel injury models: Researchers can utilize MTMR14 antibodies to track changes in MTMR14 expression following vessel injury. Studies have shown that neointima formation and MTMR14 expression increase after vessel injury, suggesting a regulatory role . Immunohistochemistry or Western blot analyses of vessel tissues at various time points post-injury can reveal temporal changes in MTMR14 expression and localization.

• Protein-protein interaction studies: MTMR14 has been shown to interact with PLK1 (polo-like kinase 1), leading to inhibition of MEK/ERK/AKT signaling pathways . Co-immunoprecipitation experiments using MTMR14 antibodies can help identify and confirm interaction partners in VSMCs, elucidating the molecular mechanisms by which MTMR14 regulates VSMC proliferation.

• Pathway analysis: Immunoblotting with phospho-specific antibodies against MEK, ERK, and AKT, in conjunction with MTMR14 antibodies, can reveal how MTMR14 modulates these signaling pathways in VSMCs under normal and pathological conditions .

• Quantitative assessment in genetic models: Comparative studies using tissues from wild-type, MTMR14-knockout, and MTMR14-transgenic mice can help establish causative relationships between MTMR14 expression levels and neointima formation . MTMR14 antibodies are essential for confirming the successful deletion or overexpression of the protein in these genetic models.

What methodological approaches can be used to study MTMR14's role in metabolism and inflammation?

To investigate MTMR14's role in metabolism and inflammation, researchers can employ several methodological approaches utilizing MTMR14 antibodies:

• Tissue-specific expression analysis: Western blot and immunohistochemistry using MTMR14 antibodies can be performed on metabolically relevant tissues (liver, adipose tissue, muscle) from both wild-type and MTMR14-deficient models to correlate protein expression with metabolic phenotypes .

• Temporal expression studies: Analysis of MTMR14 expression at different age points (early vs. aging) can help understand its role in age-related metabolic dysregulation. Research has shown that MTMR14 deficiency leads to late-onset inflammation and abnormal metabolism .

• Co-localization studies: Dual immunofluorescence staining with MTMR14 antibodies and markers of inflammation or metabolic pathways can reveal spatial relationships in tissues.

• Correlation with metabolic parameters: Researchers can analyze the relationship between MTMR14 expression levels (determined by antibody-based methods) and various metabolic parameters including:

  • Adipokine levels (leptin, adiponectin)

  • Inflammatory cytokines (TNF-α, IL-6, IL-1β)

  • Metabolic enzymes (PEPCK, G6P)

• Cell culture models: In vitro studies using adipocytes, hepatocytes, or myocytes with modulated MTMR14 expression can help elucidate cell-type specific roles, with MTMR14 antibodies serving as critical tools for validation.

How can phosphatase activity of MTMR14 be assessed in experimental settings?

• Immunoprecipitation-based phosphatase assays: MTMR14 antibodies can be used to immunoprecipitate the protein from cell or tissue lysates, followed by incubation of the immunoprecipitates with specific phosphoinositide substrates (PtdIns3P and PtdIns(3,5)P2) . The dephosphorylation can be measured by quantifying the release of inorganic phosphate or by analyzing the conversion of substrates to products using thin-layer chromatography or mass spectrometry.

• Structure-function analysis: Researchers can generate MTMR14 mutants with targeted mutations in the phosphatase domain, express these mutants in cell systems, and use MTMR14 antibodies to confirm expression levels before assessing functional consequences.

• Cellular phosphoinositide levels: Indirect assessment of MTMR14 phosphatase activity can be performed by measuring cellular levels of its substrates (PtdIns3P and PtdIns(3,5)P2) using specific probes or antibodies in wild-type versus MTMR14-deficient cells, with MTMR14 antibodies confirming the knockdown/knockout efficiency.

• Pharmacological inhibition studies: MTMR14 antibodies can be used to monitor protein levels in experiments involving potential inhibitors of its phosphatase activity, helping to distinguish between effects on protein expression versus enzymatic activity.

What are the optimal conditions for using MTMR14 antibodies in Western blot applications?

Based on validated protocols, the following conditions are recommended for optimal results when using MTMR14 antibodies in Western blot applications:

For optimal results, researchers should consider the following technical tips:

• Ensure complete transfer of high molecular weight proteins

• Include positive controls in each experiment

• Validate antibody specificity using knockdown/knockout samples when available

• Store antibodies according to manufacturer recommendations (typically at -20°C with 0.02% sodium azide and 50% glycerol, pH 7.3)

What are the key considerations for immunofluorescence applications with MTMR14 antibodies?

When using MTMR14 antibodies for immunofluorescence applications, researchers should consider the following key parameters for optimal results:

Additional considerations include:

• Inclusion of appropriate negative controls (primary antibody omission, isotype controls)

• Validation of specificity using siRNA knockdown or CRISPR knockout cells

• Assessment of colocalization with organelle markers to confirm subcellular distribution

• Careful optimization of image acquisition parameters to avoid saturation and accurately represent the staining pattern

How should researchers troubleshoot non-specific binding or lack of signal when using MTMR14 antibodies?

When encountering issues with MTMR14 antibody performance, researchers can implement the following troubleshooting strategies:

For non-specific binding:

• Antibody dilution optimization: Increase the dilution of the primary antibody incrementally (e.g., from 1:1000 to 1:2000 or 1:4000) to reduce non-specific binding while maintaining specific signal .

• Blocking optimization:

  • Test different blocking agents (BSA, casein, commercial blocking buffers)

  • Increase blocking time or concentration

  • Add 0.1-0.2% Tween-20 to washing buffers to reduce hydrophobic interactions

• Sample preparation improvements:

  • Ensure complete lysis and proper protein denaturation

  • Consider using different lysis buffers with appropriate protease inhibitors

  • For tissues, optimize extraction protocols to ensure complete solubilization

• Validation with controls:

  • Use MTMR14 knockout/knockdown samples to identify specific versus non-specific bands

  • Pre-adsorb antibody with recombinant MTMR14 protein to confirm specificity

For lack of signal:

• Sample considerations:

  • Verify MTMR14 expression in the sample type being tested

  • Increase protein loading (50-100 μg per lane)

  • Check for protein degradation during sample preparation

• Detection sensitivity:

  • Switch to a more sensitive detection system (enhanced chemiluminescence plus or super signal)

  • Increase exposure time incrementally

  • Consider signal amplification systems

• Antibody handling:

  • Avoid repeated freeze-thaw cycles of the antibody

  • Ensure proper storage conditions (typically -20°C with glycerol)

  • Check antibody expiration date

• Epitope accessibility:

  • For fixed samples, test different fixation and antigen retrieval methods

  • For Western blots, ensure efficient transfer of proteins to the membrane

How does MTMR14 expression correlate with vascular pathologies, and what implications does this have for therapeutic development?

Research has revealed significant correlations between MTMR14 expression and vascular pathologies, particularly in the context of restenosis and neointima formation:

• Expression patterns in vascular injury: Studies have demonstrated that neointima formation and MTMR14 expression increase following vessel injury . This upregulation suggests a compensatory mechanism attempting to control the pathological process.

• Functional consequences of expression modulation:

  • MTMR14 deficiency accelerates neointima formation and promotes vascular smooth muscle cell (VSMC) proliferation after injury

  • Conversely, MTMR14 overexpression remarkably attenuates neointima formation and VSMC proliferation

• Molecular mechanisms: Mechanistically, MTMR14 suppresses the activation of PLK1 (polo-like kinase 1) through direct protein interaction. This interaction inhibits the activation of downstream signaling cascades including MEK/ERK/AKT pathways, ultimately preventing VSMC proliferation and migration from the medial to the intimal layer of blood vessels .

Therapeutic implications:

The identification of MTMR14 as an inhibitor of VSMC proliferation establishes it as a potential therapeutic target for restenosis, a common complication following vascular interventions . Several approaches could be explored:

• Pharmacological enhancement: Development of small molecules that enhance MTMR14 expression or activity could potentially reduce neointima formation following angioplasty or stent placement.

• Gene therapy approaches: Local delivery of MTMR14 expression vectors at sites of vascular intervention could provide targeted enhancement of its protective effects.

• Targeted therapies: Development of drugs that mimic MTMR14's interaction with PLK1, specifically blocking this pathway in VSMCs.

• Biomarker potential: MTMR14 expression levels could serve as prognostic indicators for restenosis risk following vascular interventions.

These findings suggest that MTMR14-targeted therapies may represent a novel approach to addressing restenosis, which remains "one of the main bottlenecks in restricting the further development of cardiovascular interventional therapy" .

What is known about the relationship between MTMR14 and metabolic regulation based on current research?

Current research has established several important connections between MTMR14 and metabolic regulation, primarily through studies of MTMR14 knockout (KO) mice:

• Body weight regulation: MTMR14 KO mice gain weight earlier than their wild-type (WT) littermates, suggesting a role for this protein in body weight homeostasis and metabolism .

• Age-dependent metabolic dysregulation: The metabolic phenotype associated with MTMR14 deficiency appears to be age-dependent, with more pronounced effects observed in aged mice (≥18 weeks) .

• Tissue-specific metabolic gene expression:

  In liver tissue:

  • Adipokine mRNA levels (including leptin) are dramatically increased in MTMR14 KO mice

  • PEPCK and G6P, insulin-responsive enzymes involved in hepatic glyceroneogenesis, show significantly higher expression in MTMR14 KO mice at 18 weeks, with sustained elevation at 34 weeks

  In systemic circulation:

  • Plasma leptin levels are elevated in aged MTMR14 KO mice

  • Plasma adiponectin shows significant reduction in aged MTMR14 KO mice compared to WT littermates

• Inflammation-metabolism interface: MTMR14 deficiency leads to dysregulation of inflammatory markers in metabolically active tissues:

  • TNF-α and IL-6 mRNA levels increase in the livers of MTMR14 KO mice even at an early age

  • IL-1β and MCP-1 mRNA levels decrease in aged MTMR14 KO mice

  • Circulating inflammatory cytokines (TNF-α and IL-6) are dramatically increased in aged MTMR14 KO mice

These findings suggest that MTMR14 plays a complex role in metabolic regulation, potentially serving as a linker between phosphoinositide signaling, inflammatory pathways, and metabolic homeostasis. The age-dependent nature of these effects indicates that MTMR14 may be particularly important in preventing metabolic dysregulation during aging. Further research is needed to elucidate the precise molecular mechanisms by which MTMR14 influences metabolism and to determine if similar metabolic functions are conserved in humans.

How do the functional roles of MTMR14 relate to its phosphatase activity against specific phosphoinositide substrates?

The functional roles of MTMR14 in various biological processes are intricately linked to its phosphatase activity against specific phosphoinositide substrates:

• Substrate specificity: MTMR14 functions as a lipid phosphatase that efficiently dephosphorylates phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) . Importantly, it shows high substrate selectivity, remaining inactive toward other phosphoinositides including PtdIns4P, PtdIns(3,4)P2, PtdIns(4,5)P2, and PtdIns(3,4,5)P3 .

• Phosphoinositide signaling in vascular biology: PtdIns3P and PtdIns(3,5)P2 are critical signaling molecules involved in multiple cellular processes. In vascular smooth muscle cells (VSMCs), these phosphoinositides likely contribute to signaling cascades that regulate proliferation and migration. By dephosphorylating these substrates, MTMR14 may modulate these signaling pathways, explaining its observed role in preventing neointima formation and VSMC proliferation .

• Metabolic regulation: Phosphoinositides serve as important second messengers in insulin signaling and other metabolic pathways. The dephosphorylation of specific phosphoinositides by MTMR14 could influence key metabolic processes, potentially explaining the metabolic phenotypes observed in MTMR14 knockout mice, including early weight gain and dysregulation of metabolic genes in the liver .

• Mechanistic connection to PLK1 signaling: Research has demonstrated that MTMR14 suppresses the activation of PLK1 (polo-like kinase 1) through direct protein interaction, which consequently inhibits the MEK/ERK/AKT signaling cascade . This interaction may be dependent on or influenced by MTMR14's phosphatase activity, as phosphoinositides can regulate protein localization and complex formation.

• Regulatory mechanisms: As with other members of the myotubularin family, MTMR14's phosphatase activity may be regulated through protein-protein interactions. The MTM1/MTMRs family is characterized by associations between catalytically inactive and active members, which play important regulatory roles . Understanding these regulatory interactions could provide insights into how MTMR14's phosphatase activity is controlled in various cellular contexts.

While the precise molecular mechanisms linking MTMR14's phosphatase activity to its diverse functional roles are still being elucidated, the available evidence suggests that its ability to modulate specific phosphoinositide pools is central to its biological functions. Future research utilizing point mutations that specifically abolish phosphatase activity while preserving protein structure will be valuable in distinguishing phosphatase-dependent and phosphatase-independent functions of MTMR14.

What emerging techniques might enhance the utility of MTMR14 antibodies in studying phosphoinositide signaling?

Several emerging techniques hold promise for enhancing the utility of MTMR14 antibodies in studying phosphoinositide signaling:

• Proximity labeling approaches: Techniques such as BioID or APEX2 could be combined with MTMR14 antibodies to identify proteins that interact with MTMR14 in specific subcellular compartments. These approaches involve expressing MTMR14 fused to a promiscuous biotin ligase, followed by streptavidin pulldown and identification of biotinylated proteins that were in proximity to MTMR14. Antibodies are crucial for validating the expression of the fusion protein and confirming the identified interactions.

• Super-resolution microscopy: Advanced imaging techniques like STORM, PALM, or STED microscopy, when used with highly specific MTMR14 antibodies, can provide nanoscale localization of MTMR14 relative to its substrates and interacting partners. This could reveal previously undetected spatial relationships between MTMR14 and phosphoinositide pools.

• Live-cell phosphoinositide sensors: Combining phosphoinositide-specific biosensors (for PtdIns3P and PtdIns(3,5)P2) with modulation of MTMR14 expression could enable real-time visualization of how MTMR14 affects its substrate pools in living cells. MTMR14 antibodies would be essential for confirming expression levels in these experiments.

• Mass spectrometry-based phosphoproteomics: This approach could identify downstream effectors of MTMR14-mediated phosphoinositide signaling by comparing the phosphoproteome in MTMR14-deficient versus wild-type cells. MTMR14 antibodies would be crucial for validating the experimental system.

• CRISPR-based genomic screens: These screens could identify genes that synergize with or antagonize MTMR14 function. MTMR14 antibodies would be needed to confirm protein expression in the screen components and validate hits.

• Single-cell analysis techniques: Combining MTMR14 immunostaining with single-cell RNA-seq or mass cytometry could reveal cell-to-cell variability in MTMR14 expression and its correlation with cellular phenotypes or signaling states.

What are the critical considerations for designing experiments to investigate MTMR14's role in disease models?

When designing experiments to investigate MTMR14's role in disease models, researchers should consider the following critical factors:

• Model selection and validation:

  • Choose appropriate disease models based on MTMR14's known functions in vascular biology, metabolism, and inflammation

  • For vascular disease models, consider wire injury, balloon injury, or carotid ligation models that have been validated for studying neointima formation

  • For metabolic disease models, consider high-fat diet challenges in MTMR14 knockout mice to exacerbate the metabolic phenotype

  • Validate MTMR14 expression patterns in the selected models using antibodies with confirmed specificity

• Genetic approach considerations:

  • Consider tissue-specific conditional knockout models (e.g., SMC-specific conditional MTMR14-knockout mice for vascular studies)

  • Include both knockout and overexpression models to assess loss-of-function and gain-of-function effects

  • Consider the timing of genetic manipulation (developmental vs. adult) as MTMR14's functions appear to have age-dependent components

  • Use appropriate controls (littermates with the same genetic background)

• Experimental design factors:

  • Include time-course analyses to capture dynamic changes in MTMR14 expression and function

  • Design experiments to distinguish between direct and indirect effects of MTMR14 modulation

  • Consider compensatory mechanisms that may mask phenotypes in chronic knockout models

  • Include both male and female animals to identify potential sex-specific effects

• Analytical approaches:

  • Employ a multi-omics approach (transcriptomics, proteomics, metabolomics) to capture the full spectrum of MTMR14-dependent changes

  • Use appropriate statistical methods for data analysis, considering factors like sample size, variability, and multiple comparisons

  • Include mechanistic studies to connect phenotypic observations to molecular mechanisms

  • Consider pathway analysis to place MTMR14-dependent changes in a broader biological context

• Translational considerations:

  • Assess the relevance of findings in animal models to human disease

  • Consider pharmacological approaches to modulate MTMR14 function in addition to genetic approaches

  • Evaluate potential biomarker applications based on MTMR14 expression or activity

By carefully considering these factors, researchers can design robust experiments to elucidate MTMR14's role in disease models and potentially identify novel therapeutic approaches.

How can researchers address age-dependent variations in MTMR14 expression when designing experiments?

The observed age-dependent variations in MTMR14 expression and its associated phenotypes present important challenges for experimental design. Researchers can implement the following strategies to address these variables:

• Age-matched experimental groups: Always use age-matched controls and experimental subjects, with precise documentation of age (preferably in weeks rather than months) . Research has shown significant differences in MTMR14-related phenotypes between mice at 4 weeks, 18 weeks, and 34 weeks of age.

• Time-course experimental design: Design experiments that track MTMR14 expression and related phenotypes across multiple age points to capture the dynamic nature of its regulation. Based on previous studies, critical time points to consider include:

  • Early age (4-8 weeks)

  • Middle age (16-20 weeks)

  • Advanced age (30+ weeks)

• Standardized analysis methods: Develop and consistently apply standardized methods for measuring MTMR14 expression levels:

  • For protein detection, use validated MTMR14 antibodies with established dilutions and protocols

  • Include internal loading controls appropriate for the tissue type being analyzed

  • Consider normalization strategies that account for age-related changes in reference proteins

• Multi-parameter assessment: Simultaneously measure MTMR14 expression alongside relevant age-dependent markers:

  • Inflammatory cytokines (TNF-α, IL-6)

  • Metabolic markers (adipokines, insulin signaling components)

  • Tissue-specific markers of aging

• Statistical considerations:

  • Power analyses should account for increased variability in aged animals

  • Use statistical methods that can account for age as a continuous variable when appropriate

  • Consider increased sample sizes for aged cohorts to compensate for greater biological variability

• Technical validation: Validate antibody performance across different age groups, as protein modifications or interacting partners that vary with age could affect epitope accessibility or antibody binding.

By implementing these strategies, researchers can more effectively address age-dependent variations in MTMR14 expression and develop more robust experimental designs for studying its functions across the lifespan.

What quality control measures should be implemented when using MTMR14 antibodies in quantitative applications?

To ensure reliability and reproducibility when using MTMR14 antibodies in quantitative applications, researchers should implement the following quality control measures:

• Antibody validation:

  • Verify antibody specificity using positive controls (tissues/cells known to express MTMR14) and negative controls (MTMR14 knockout or knockdown samples)

  • Conduct peptide competition assays to confirm binding specificity

  • Compare results from multiple MTMR14 antibodies targeting different epitopes when possible

• Standard curve development:

  • For quantitative Western blot, develop standard curves using recombinant MTMR14 protein

  • Determine the linear range of detection for each application and antibody

  • Validate that signal intensity correlates linearly with protein concentration within the working range

• Technical replication:

  • Include technical replicates (minimum of three) for each biological sample

  • Calculate coefficient of variation between replicates, with acceptance criteria typically <15%

  • Establish protocol for handling outliers prior to experiment initiation

• Normalization strategies:

  • For Western blot, normalize to appropriate loading controls verified to be stable under experimental conditions

  • For immunofluorescence quantification, use reference markers or total cell number for normalization

  • Validate that normalization controls are not affected by experimental conditions

• Calibration and instrument qualification:

  • Regularly calibrate imaging equipment (Western blot imagers, microscopes)

  • Establish standard operating procedures for image acquisition to ensure consistency

  • Use intensity calibration standards appropriate for the detection method

• Documentation and reporting:

  • Document lot numbers and validation data for all antibodies used

  • Record detailed protocols including antibody dilutions, incubation times, and washing conditions

  • Report all quality control measures in publications or technical reports

• Application-specific controls:

  For Western blot quantification:

  • Include molecular weight markers to confirm target band size (60-72 kDa for MTMR14)

  • Run gradient gels when comparing samples with potentially different post-translational modifications

  • Include positive control samples on each gel for inter-gel normalization

  For immunohistochemistry/immunofluorescence quantification:

  • Include isotype controls to assess non-specific binding

  • Use blocking peptides to confirm staining specificity

  • Establish consistent thresholds for positive staining