LMOD1 Antibody

What is the tissue expression pattern of LMOD1 and how does this affect antibody selection?

LMOD1 exhibits a highly restricted expression pattern primarily in smooth muscle cells (SMCs). Immunohistochemistry and immunoblotting have demonstrated that LMOD1 is predominantly expressed in:

• Blood vessel walls (vascular smooth muscle cells)

• Visceral organs with smooth muscle components

• Gastrointestinal tract walls

• Urinary bladder (detrusor muscle)

When selecting an LMOD1 antibody, researchers should consider this tissue distribution. For brain studies, it's critical to recognize that LMOD1 is primarily localized to smooth muscle cells of cerebral blood vessels rather than neurons or glia . This has significant implications for experimental design and interpretation, particularly when studying potential neurological conditions like nodding syndrome .

What are the recommended applications and dilutions for LMOD1 antibodies?

LMOD1 antibodies can be used for multiple experimental techniques with specific dilution recommendations:

For optimal results, antigen retrieval methods may significantly impact staining outcomes:

• For IHC: Use TE buffer pH 9.0 or alternatively citrate buffer pH 6.0

• Always perform antibody titration in your specific experimental system to determine optimal conditions

How should researchers select between different LMOD1 antibody epitopes?

Selection of LMOD1 antibodies targeting different epitopes is critical as they can produce different staining patterns:

• N-terminal-targeting antibodies (e.g., PA5-44224):

  • May cross-react with tropomodulin due to sequence homology at the N-terminus

  • Show immunoreactivity in Purkinje cell membranes and C. elegans body wall

  • Suitable for detecting potential cross-reactivity with tropomodulin-like proteins

• Full-length protein antibodies (e.g., NBP1-89398):

  • More specific to LMOD1

  • Do not typically stain Purkinje cells or C. elegans

  • Recommended for applications requiring high specificity to LMOD1

When studying potential molecular mimicry or cross-reactivity in autoimmune conditions, researchers should consider using both antibody types to distinguish between true LMOD1 expression and potential cross-reactivity with homologous proteins like tropomodulin .

What is the molecular weight profile of LMOD1 in Western blot analysis?

LMOD1 protein exhibits variable molecular weights in Western blot analysis:

The discrepancy between calculated and observed molecular weights may be attributed to:

• Post-translational modifications

• Tissue-specific isoforms

• Protein-protein interactions affecting migration

• Different antibody epitopes recognizing different forms of the protein

When performing Western blot analysis for LMOD1, researchers should include appropriate molecular weight markers and positive control samples (e.g., smooth muscle-containing tissues) to accurately identify LMOD1-specific bands .

How can researchers distinguish between LMOD1 and its homologous proteins in immunostaining experiments?

Distinguishing LMOD1 from its homologs (LMOD2, LMOD3) and related tropomodulin proteins requires careful experimental design:

• Antibody selection strategy:

  • Use antibodies targeting non-conserved regions of LMOD1

  • Perform parallel staining with antibodies to LMOD2/LMOD3 and tropomodulins

  • Include appropriate tissue controls (LMOD1: smooth muscle; LMOD2/3: cardiac/skeletal muscle)

• Validation approaches:

  • Preabsorption controls with purified antigens

  • Knockout/knockdown validation systems

  • Peptide competition assays

• Tissue-specific expression patterns:

  • LMOD1: Primarily smooth muscle tissues

  • LMOD2/LMOD3: Predominantly skeletal and cardiac muscle with very low expression in smooth muscle tissues

Research by Kodja et al. demonstrated that sequence homology between LMOD1 and tropomodulin, particularly at the N-terminus, caused cross-reactivity of certain LMOD1 antibodies with tropomodulin in Purkinje cells and C. elegans . This highlights the importance of using multiple antibodies targeting different epitopes to confirm specificity.

What are the critical methodological considerations when using LMOD1 antibodies in brain tissue analysis?

When studying LMOD1 in brain tissue, researchers should consider several technical factors:

• Cellular localization patterns:

  • LMOD1 is primarily expressed in smooth muscle cells of cerebral blood vessels, not in neurons or glia

  • Any apparent neuronal staining should be carefully validated to rule out cross-reactivity with tropomodulin or other actin-binding proteins

• Technical approaches for validation:

  • Compare staining patterns using antibodies targeting different LMOD1 epitopes

  • Co-staining with smooth muscle markers (e.g., ACTA2) to confirm vascular localization

  • Include negative controls (primary antibody omission)

  • Include tissues from LMOD1 knockout models as specificity controls

• Developmental considerations:

  • Examine LMOD1 expression across developmental stages (neonate to adult)

  • Age-appropriate controls are essential when studying developmental disorders

The study by Kodja et al. examining LMOD1 in nodding syndrome patients found that LMOD1 was not expressed in neurons or Purkinje cells when using full-length LMOD1 antibodies, contradicting earlier findings and suggesting cross-reactivity in previous studies .

How should researchers design experiments to study LMOD1's role in smooth muscle cell contractility?

To investigate LMOD1's function in smooth muscle contractility:

• In vitro experimental approaches:

  • LMOD1 knockdown/knockout in primary human coronary artery smooth muscle cells (HCASMC)

  • Assessment of proliferation, migration, and contractility following LMOD1 depletion

  • Actin cytoskeleton visualization using phalloidin staining

  • Contraction assays using collagen gel matrices

• In vivo model systems:

  • CRISPR-Cas9 engineered LMOD1 knockout mice

  • Tissue-specific conditional knockout models

  • Transgenic mice with tagged LMOD1 for lineage tracing

• Functional readouts:

  • Microscopic analysis of smooth muscle layers (thickness, organization)

  • Ki67 staining to assess proliferation changes

  • Ultrastructural analysis of actin filaments by electron microscopy

  • Organ-specific functional tests (e.g., bladder pressure measurements)

Research by Halim et al. demonstrated that LMOD1 knockout in mice results in megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS), characterized by distended bladder and impaired smooth muscle contractility . This phenotype matches that observed in a human patient with a homozygous nonsense mutation in LMOD1 .

What experimental controls are essential for validating LMOD1 antibody specificity?

To ensure reliable results with LMOD1 antibodies, researchers should implement comprehensive validation controls:

• Genetic validation controls:

  • Tissues from LMOD1 knockout/knockdown models

  • siRNA-treated cell cultures with confirmed LMOD1 depletion

  • Comparison with established LMOD1 expression patterns

• Technical validation controls:

  • Antibody preabsorption with purified LMOD1 antigen

  • Omission of primary antibody

  • Isotype controls (e.g., rabbit IgG at equivalent concentration)

  • Peptide competition assays

• Cross-reactivity assessment:

  • Parallel testing with multiple antibodies targeting different LMOD1 epitopes

  • Testing in tissues known to lack LMOD1 expression

  • Western blot correlation with immunohistochemical findings

In their study of nodding syndrome, Kodja et al. demonstrated the importance of these controls by showing that antibodies to the N-terminal region of LMOD1 produced staining in Purkinje cells that was likely due to cross-reactivity with tropomodulin, while antibodies to the full-length protein showed no such staining .

How can researchers investigate the regulatory mechanisms controlling LMOD1 expression?

To study the transcriptional regulation of LMOD1:

• Promoter analysis approaches:

  • Cloning of LMOD1 promoter regions into reporter constructs

  • Site-directed mutagenesis of regulatory elements (e.g., CArG boxes)

  • Chromatin immunoprecipitation (ChIP) assays for transcription factor binding

  • Transgenic mouse studies with wild-type and mutant promoters

• Key regulatory elements to examine:

  • Two conserved CArG boxes in the LMOD1 promoter (-291 to +249 bp region)

  • Serum response factor (SRF) binding sites

  • FOXO3 binding motifs altered by CAD-associated variants

• Experimental readouts:

  • Luciferase reporter activity in response to transcription factors

  • Expression quantitative trait loci (eQTL) analysis in vascular tissues

  • Effects of transcription factor knockdown on LMOD1 expression

Research by Nanda et al. revealed that LMOD1 is a target gene of the serum response factor (SRF)/myocardin (MYOCD) transcriptional switch, with its expression dependent on two functional CArG elements in the promoter region . Additionally, Zhao et al. identified a non-coding regulatory variant (rs34091558) as the top regulatory variant for LMOD1 expression in vascular tissues, which disrupts a conserved FOXO3 binding motif .

What are common troubleshooting strategies for weak or nonspecific LMOD1 antibody signals?

When encountering issues with LMOD1 detection:

For IHC applications specifically, researchers should consider the following optimization steps:

• Test both TE buffer pH 9.0 and citrate buffer pH 6.0 for antigen retrieval

• Compare heat-induced versus enzymatic epitope retrieval methods

• Extend primary antibody incubation time (overnight at 4°C may yield better results)

• Include detergent (0.1-0.3% Triton X-100) to improve antibody penetration

How should researchers interpret conflicting LMOD1 antibody results between different experimental techniques?

When faced with discrepancies between different detection methods:

• Systematic evaluation approach:

  • Compare antibody characteristics (epitope, clonality, validation methods)

  • Evaluate detection sensitivity of each technique (WB vs. IHC vs. IF)

  • Consider cross-reactivity potential with related proteins

• Reconciliation strategies:

  • Use multiple antibodies targeting different epitopes

  • Confirm results with orthogonal techniques (e.g., mRNA expression, reporter assays)

  • Include appropriate positive and negative controls for each technique

  • Consider native protein conformation versus denatured states

• Case study from literature:

  • Kodja et al. observed discrepancies between antibodies targeting different regions of LMOD1

  • N-terminal antibodies showed staining in Purkinje cells while full-length protein antibodies did not

  • These differences were attributed to cross-reactivity with tropomodulin at the N-terminus

  • This example demonstrates the importance of using multiple antibodies and careful validation

This approach is particularly important when studying LMOD1 in the context of autoimmune conditions or when examining tissues with potential cross-reactive proteins.

How can LMOD1 antibodies be applied to study smooth muscle cell phenotypic switching?

LMOD1 serves as a marker for the differentiated smooth muscle cell (SMC) phenotype:

• Research design considerations:

  • Co-staining with other SMC markers (ACTA2, TAGLN, MYH11)

  • Assessment of LMOD1 expression changes during phenotypic modulation

  • Correlation with SMC contractile function

• Relevant disease models:

  • Atherosclerosis models (SMC lineage tracing)

  • Vascular injury models

  • Coronary artery disease (CAD) genetic association studies

• Experimental approaches:

  • SMC lineage tracing reporter mice to track LMOD1 expression during disease progression

  • Analysis of LMOD1 expression in response to growth factors/inflammatory stimuli

  • Assessment of actin cytoskeleton organization during phenotypic switching

Research by Zhao et al. demonstrated that LMOD1 plays a key role in maintaining the differentiated SMC phenotype, with LMOD1 knockdown resulting in increased proliferation and migration and decreased contractility in human coronary artery smooth muscle cells (HCASMCs) .

What is the role of LMOD1 antibodies in investigating visceral smooth muscle disorders?

LMOD1 antibodies are valuable tools for studying disorders affecting visceral smooth muscle:

• Relevant clinical conditions:

  • Megacystis microcolon intestinal hypoperistalsis syndrome (MMIHS)

  • Gastrointestinal motility disorders

  • Bladder dysfunctions

• Research applications:

  • Assessment of LMOD1 expression in patient tissues

  • Analysis of smooth muscle architecture in affected organs

  • Correlation of LMOD1 levels with disease severity

• Experimental models:

  • LMOD1 knockout mice exhibit MMIHS-like phenotypes

  • Patient-derived cells (e.g., fibroblasts from affected individuals)

  • Organ-specific functional studies (e.g., contractility assays)

Research by Halim et al. identified a homozygous nonsense mutation in LMOD1 in a child with MMIHS and demonstrated that mice with similar LMOD1 mutations exhibit comparable gastrointestinal and urinary bladder phenotypes . Histological analysis revealed thinning of detrusor muscle in the bladder and impaired smooth muscle contractility, establishing LMOD1's critical role in visceral smooth muscle function .

How should researchers approach the study of LMOD1 in potential autoimmune conditions?

When investigating LMOD1's role in autoimmune diseases:

• Experimental design considerations:

  • Analysis of autoantibody binding to different LMOD1 epitopes

  • Assessment of cross-reactivity with homologous proteins

  • Careful selection of LMOD1 antibodies to avoid conflating true autoantibodies with technical artifacts

• Critical technical approaches:

  • Parallel detection with multiple anti-LMOD1 antibodies targeting different regions

  • Western blot validation of sera reactivity

  • Pre-absorption controls with purified antigen

  • Competition assays between patient sera and commercial antibodies

• Relevant disease contexts:

  • Nodding syndrome (proposed autoimmunity to LMOD1)

  • Hashimoto's thyroiditis (LMOD1 was initially identified as an autoantigen)

  • Thyroid-associated ophthalmopathy

Research by Kodja et al. challenged the hypothesis that nodding syndrome is caused by autoantibodies to LMOD1 cross-reacting with Onchocerca volvulus tropomyosin-like proteins . Their findings demonstrated that LMOD1 is not widely expressed in neurons as previously claimed, suggesting that if autoimmunity to LMOD1 exists, it would target vascular smooth muscle rather than neurons .

What emerging applications of LMOD1 antibodies should researchers consider?

Several innovative applications for LMOD1 antibodies hold promise for future research:

• Single-cell analysis approaches:

  • Single-cell immunostaining to detect LMOD1 heterogeneity within smooth muscle populations

  • Correlation with single-cell transcriptomics data

  • Analysis of LMOD1 expression during smooth muscle cell differentiation

• Advanced imaging applications:

  • Super-resolution microscopy to examine LMOD1's interaction with the actin cytoskeleton

  • Live-cell imaging with fluorescently tagged LMOD1 antibody fragments

  • Expansion microscopy for improved visualization of subcellular LMOD1 localization

• Therapeutic monitoring applications:

  • Assessment of LMOD1 expression as a biomarker for smooth muscle-related disorders

  • Monitoring LMOD1 levels during therapeutic interventions targeting smooth muscle function

  • Development of non-invasive methods to detect LMOD1 dysregulation

These emerging applications could significantly advance our understanding of LMOD1's role in smooth muscle biology and disease pathogenesis.