LMO7 Antibody

What is LMO7 and why is it significant for research?

LMO7 is a multifunctional PDZ-LIM protein that interacts with various molecular partners and is found in several intracellular locations. The LMO7 gene is located on chromosome 13q14.11 in humans, encoding a protein that contains multiple domains including LIM domains (protein-protein interaction motifs), PDZ domains (interaction modules that bind to specific protein sequences), and CH domains (actin-binding motifs) . LMO7 has garnered significant research interest due to its involvement in diverse physiological processes including neuronal development, cardiovascular health, and cancer pathogenesis . For researchers, LMO7 represents an important target for studying signaling pathways related to cell growth, differentiation, cytoskeletal organization, and particularly cancer development.

Where is LMO7 protein predominantly expressed in normal tissues?

LMO7 protein is predominantly expressed in cardiac and skeletal muscle tissues . Immunohistochemistry experiments have successfully detected positive LMO7 expression in mouse heart tissue and mouse skeletal muscle tissue . Additionally, Western blot analysis has confirmed LMO7 expression in A549 cells, HeLa cells, and mouse lung tissue . In vascular tissue, LMO7 is expressed at modest levels in uninjured vessels but becomes highly induced following injury, with expression peaking around day 10 post-injury while remaining elevated through 28 days . This tissue-specific expression pattern is important to consider when designing experiments and selecting appropriate positive control tissues for antibody validation.

What is the molecular weight of LMO7 protein, and why might it vary in experiments?

The calculated molecular weight of LMO7 protein is 193 kDa, but the observed molecular weight in experimental contexts typically ranges from 140-160 kDa . This discrepancy between calculated and observed molecular weights can be attributed to several factors:

• Post-translational modifications (phosphorylation, glycosylation, etc.)

• Alternative splicing generating different isoforms

• Proteolytic processing of the full-length protein

• The presence of intrinsically disordered regions affecting migration patterns

Human LMO7 has at least 27 different predicted transcripts, nine of which are non-coding, while the others produce protein variants with potentially different molecular weights . Researchers should be aware of these variations when interpreting Western blot results and may need to validate which isoform they are detecting in their experimental system.

What are the recommended applications and dilutions for LMO7 antibodies?

Based on validated antibody data, LMO7 antibodies can be successfully applied in several experimental contexts:

For immunohistochemistry applications, antigen retrieval with TE buffer pH 9.0 is suggested, though citrate buffer pH 6.0 may serve as an alternative . It is strongly recommended that researchers titrate the antibody in their specific testing system and sample type to obtain optimal results, as antibody performance can be sample-dependent.

How should I optimize Western blot protocols for detecting LMO7?

When optimizing Western blot protocols for LMO7 detection, consider the following methodological approaches:

• Sample preparation: Use strong lysis buffers (RIPA buffer with protease inhibitors) to ensure complete extraction of membrane-associated and nuclear LMO7 protein.

• Gel percentage: Due to the high molecular weight of LMO7 (observed at 140-160 kDa), use lower percentage gels (6-8% acrylamide) to achieve better separation of high molecular weight proteins.

• Transfer conditions: Employ longer transfer times (overnight at low voltage or 2+ hours at higher voltage) with added SDS in the transfer buffer (0.1%) to facilitate transfer of large proteins.

• Blocking and antibody incubation:

  • Use 5% non-fat dry milk or BSA in TBST for blocking (1-2 hours at room temperature)

  • Incubate with primary antibody overnight at 4°C (using the recommended dilution of 1:2000-1:10000)

  • Wash thoroughly (4-5 times for 5-10 minutes each) before secondary antibody incubation

• Controls: Include positive controls from tissues known to express LMO7 (heart, skeletal muscle, or lung tissue) .

• Expected bands: Be prepared to observe bands between 140-160 kDa, and potentially additional bands representing alternative isoforms .

When troubleshooting, remember that due to LMO7's large size and potential post-translational modifications, transfer efficiency and detection sensitivity may require optimization.

What are the best approaches for immunohistochemical detection of LMO7?

For optimal immunohistochemical detection of LMO7:

• Fixation: Use 10% neutral buffered formalin fixation for 24-48 hours, avoiding over-fixation.

• Antigen retrieval: Heat-induced epitope retrieval with TE buffer pH 9.0 is recommended as the primary method, with citrate buffer pH 6.0 as an alternative .

• Antibody dilution: Start with a mid-range dilution (1:200) from the recommended range (1:50-1:500) and adjust based on signal intensity and background.

• Detection system: Use a high-sensitivity detection system, especially for tissues with lower LMO7 expression levels.

• Positive control tissues: Include mouse heart tissue and skeletal muscle tissue as positive controls to validate staining specificity .

• Negative controls: Include primary antibody omission controls and, if possible, tissues from LMO7 knockout models.

When analyzing results, pay attention to the expected subcellular localization patterns, as LMO7 can be found in multiple cellular compartments depending on the cell type and condition being studied.

How is LMO7 expression altered in different cancer types?

LMO7 expression shows variability across cancer types, functioning as both a tumor suppressor and potential oncogene depending on the cancer context:

• Upregulation in cancers:

  • Colorectal cancer: LMO7 transcription is upregulated in colorectal cancer compared to normal mucosa, with significantly higher expression in tumors carrying p53 mutations .

  • Various other cancers show LMO7 upregulation according to GEPIA 2 database analysis .

• Downregulation in cancers:

  • Lung adenocarcinoma: LMO7 acts as a tumor suppressor with LMO7-deficient mice developing lung adenocarcinoma at old age .

  • LMO7 and LIMCH1 are highly expressed in normal lung tissue but reduced in malignant lung tissue .

• Prognostic significance:

  • In LRIG1-positive tumors, LMO7 immunoreactivity predicts poor prognosis .

  • Expression patterns correlate with metastatic potential in some cancers, with higher LMO7 expression observed in highly metastatic cells compared to low metastatic cells .

Researchers studying LMO7 in cancer contexts should consider this tissue-specific and context-dependent expression pattern when designing experiments and interpreting results. Comparing expression across multiple cancer types using the same detection methods is recommended to understand these differential patterns.

What mechanisms underlie LMO7's role in cancer progression?

LMO7 influences cancer progression through several key mechanisms:

• Epithelial-Mesenchymal Transition (EMT) regulation:

  • LMO7 promotes EMT by regulating transcription factors such as snail, slug, and ZEB1

  • This regulation facilitates the loss of cell-cell adhesion and acquisition of invasive properties by cancer cells

  • Through activation of these transcription factors, LMO7 enables reprogramming of target genes involved in EMT

• TGF-β signaling modulation:

  • LMO7 functions as a negative feedback regulator of TGF-β signaling

  • Loss of LMO7 enhances TGF-β signaling by upregulating TGF-β1 mRNA, TGF-β protein, and downstream effectors including Smad3 phosphorylation

  • Mechanistically, LMO7's LIM domain interacts with transcription factors c-Fos and c-Jun, promoting their degradation and interrupting TGF-β autoinduction

• Immune evasion mechanisms:

  • In pancreatic ductal adenocarcinoma (PDAC), LMO7 drives immune evasion through regulatory T cell (Treg) enrichment

  • LMO7 promotes Treg cell differentiation and chemotaxis while inhibiting CD8+ T cells and natural killer cell cytotoxicity

  • Mechanistically, LMO7 binds and promotes the ubiquitination and degradation of Foxp1, which negatively regulates TGF-β and CCL5 expression

Understanding these mechanistic pathways provides potential targets for therapeutic intervention in cancers where LMO7 contributes to progression and immune evasion.

How do different LMO7 isoforms affect experimental results?

LMO7 genes undergo extensive alternative splicing, generating multiple isoforms that can significantly impact experimental results:

• Isoform diversity across species:

  • Humans: 27 different predicted transcripts (18 protein-coding, 9 non-coding)

  • Mice: 16 transcripts (7 protein-coding, 9 non-coding)

  • Chicken: 8 protein-coding transcripts

  • Xenopus: 6 protein-coding transcripts

  • Zebrafish: Multiple transcripts across two LMO7 genes (lmo7a and lmo7b)

• Structural and functional differences:

  • Human LMO7 isoform 3 lacks residues 359 to 690, which contain two main antiparallel α-helices and four short α-helices along with disordered regions

  • All five human LMO7 isoforms share the main long disordered regions, the PDZ domain, and the predicted nuclear localization signal (NLS)

• Experimental considerations:

  • Antibodies may detect multiple isoforms depending on the epitope location

  • PCR primers should be designed with isoform specificity in mind

  • When performing functional studies, researchers should verify which isoform(s) they are working with

To address isoform variability, researchers should:

• Use isoform-specific primers when possible

• Consider epitope locations when selecting antibodies

• Validate which isoforms are expressed in their experimental system using sequencing or isoform-specific detection methods

• Report which isoforms were studied in publications to improve reproducibility

What approaches should be used to study LMO7 protein-protein interactions?

Given LMO7's role as a multidomain protein that interacts with various partners, several methodological approaches are recommended:

• Co-immunoprecipitation (Co-IP):

  • Use antibodies against LMO7 to pull down protein complexes

  • Consider using domain-specific antibodies to determine which domains mediate specific interactions

  • Include appropriate controls (IgG controls, input controls) and validate interaction specificity

  • Example: Co-IP confirmed interaction between LRIG3 and LMO7, showing co-localization and co-immunoprecipitation

• Proximity ligation assays (PLA):

  • Useful for detecting protein-protein interactions in situ with subcellular resolution

  • Particularly valuable for LMO7 given its presence in multiple cellular compartments

• Domain-based interaction mapping:

  • Generate constructs expressing individual domains (LIM, PDZ, CH domains)

  • Use these in pull-down assays to identify domain-specific interactions

  • Research has shown the LIM domain of LMO7 interacts with c-Fos and c-Jun transcription factors

• Functional validation:

  • After identifying interactions, validate their functional significance through mutagenesis studies

  • Target key residues within interaction domains

  • Assess the effect of mutations on downstream signaling pathways like TGF-β signaling

• Computational analysis:

  • Leverage information about LMO7's intrinsically disordered regions (residues 321-344, 753-929, 938-1042, and 1236-1603) when designing interaction studies

  • These disordered regions often mediate protein-protein interactions and may be critical for LMO7's diverse functions

When reporting interaction studies, researchers should specify the isoform used, the cellular context, and validation methods to ensure reproducibility.

What are emerging therapeutic targets associated with the LMO7-Foxp1-TGF-β/CCL5 axis?

Recent research has identified the LMO7-Foxp1-TGF-β/CCL5 signaling axis as a promising therapeutic target, particularly in pancreatic ductal adenocarcinoma (PDAC):

• Mechanistic basis for targeting:

  • LMO7, through its LIM domain, directly binds and promotes the ubiquitination and degradation of Foxp1

  • Foxp1 negatively regulates TGF-β and CCL5 expression by binding to specific regulatory sites

  • Elevated TGF-β and CCL5 levels contribute to regulatory T cell enrichment, inducing immune evasion in PDAC

• Therapeutic approaches:

  • Combined treatment with TGF-β/CCL5 neutralizing antibodies along with LMO7 inhibition effectively reverses immune evasion in PDAC

  • This combined approach activates immune responses and prolonged mouse survival in experimental models

  • Targeting specific domains (particularly the LIM domain) of LMO7 may disrupt its interaction with Foxp1

• Research methodology considerations:

  • When studying this axis, researchers should monitor:

    • LMO7 and Foxp1 expression levels

    • TGF-β and CCL5 production

    • Regulatory T cell infiltration and function

    • CD8+ T cell and NK cell cytotoxicity

• Translational potential:

  • This axis represents a novel immunotherapeutic strategy for PDAC, an immunologically "cold" tumor with limited responsiveness to current immunotherapies

  • The heterogeneity of PDAC necessitates characterizing patient-specific immune microenvironment profiles to identify those who may benefit from targeting this pathway

Researchers exploring this therapeutic axis should employ combinatorial approaches that target multiple aspects of the pathway simultaneously while monitoring immune cell populations and their functional status within the tumor microenvironment.

How can I troubleshoot weak or absent LMO7 signal in Western blots?

When faced with weak or absent LMO7 signal in Western blot applications, consider these methodological solutions:

• Sample preparation issues:

  • Ensure complete protein extraction using stronger lysis buffers (RIPA with 1% SDS)

  • Add protease inhibitors to prevent degradation

  • Avoid repeated freeze-thaw cycles of protein samples

  • Confirm you're using appropriate positive control samples (A549 cells, HeLa cells, mouse lung tissue)

• Protein amount and transfer problems:

  • Increase total protein amount loaded (start with 50-80 μg for tissues with lower expression)

  • For high molecular weight proteins like LMO7 (140-160 kDa), use extended transfer times and lower percentage gels

  • Add 0.1% SDS to transfer buffer to facilitate migration of large proteins

  • Consider using PVDF membrane instead of nitrocellulose for better protein retention

• Antibody-related factors:

  • Increase primary antibody concentration (try 1:2000 if 1:10000 yields no signal)

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

  • Verify antibody storage conditions (aliquot to avoid repeated freeze-thaw cycles)

  • Check antibody expiration date and consider a new lot if necessary

• Detection system optimization:

  • Use more sensitive detection reagents (enhanced ECL substrates)

  • Increase exposure time during imaging

  • Consider alternative detection methods (fluorescent secondary antibodies may offer better sensitivity)

• Expression level considerations:

  • Remember that LMO7 expression varies by tissue and cell type, being highest in cardiac and skeletal muscle

  • In cancer contexts, expression may be upregulated or downregulated depending on the cancer type

What factors affect LMO7 detection in immunohistochemistry experiments?

Successful detection of LMO7 in immunohistochemistry experiments can be influenced by several key factors:

• Tissue preparation and fixation:

  • Overfixation can mask epitopes; limit fixation to 24-48 hours in 10% neutral buffered formalin

  • Proper tissue handling and embedding techniques are essential

  • Section thickness should be consistent (4-5 μm recommended)

• Antigen retrieval optimization:

  • LMO7 epitopes may be particularly sensitive to retrieval conditions

  • Primary recommendation: TE buffer pH 9.0

  • Alternative: citrate buffer pH 6.0

  • Optimize retrieval time and temperature (typically 95-100°C for 15-30 minutes)

• Antibody dilution and incubation:

  • Start in the middle of the recommended range (1:200) and adjust based on results

  • Extended incubation times (overnight at 4°C) may improve sensitivity

  • Use humidity chambers to prevent section drying

• Detection system considerations:

  • Polymer-based detection systems often provide better sensitivity than biotin-based systems

  • For tissues with lower expression, amplification steps may be necessary

  • Optimize chromogen development time based on signal intensity

• Counterstaining and mounting:

  • Overstaining with hematoxylin can obscure weak positive signals

  • Use appropriate mounting media that doesn't interfere with signal preservation

When troubleshooting, always include positive control tissues (mouse heart and skeletal muscle) alongside your experimental samples to confirm that technical aspects of the protocol are working properly .

How does LMO7's role in TGF-β signaling impact fibrosis research methodologies?

LMO7's function as a negative feedback regulator of TGF-β signaling presents important methodological considerations for fibrosis research:

• Experimental design considerations:

  • LMO7 expression is induced by TGF-β1 in a concentration- and time-dependent manner, with peak protein expression at 0.5 ng/ml TGF-β1 for up to 48 hours in human coronary artery smooth muscle cells

  • LMO7 induction follows the initiation of TGF-β signaling (as indicated by p-SMAD3) in vascular injury models

  • Global or smooth muscle cell-specific LMO7 deletion enhances neointimal formation, TGF-β signaling, ECM deposition, and proliferation in vascular injury models

• Methodological approaches:

  • When studying fibrotic processes, monitor both LMO7 and TGF-β pathway components (TGF-β1, p-SMAD3, CTGF) together

  • Include time course analyses to capture the temporal relationship between TGF-β signaling activation and LMO7 induction

  • Consider using LMO7 knockout/knockdown models to assess enhanced fibrotic responses

• Technical recommendations:

  • For in vitro studies, standardize TGF-β1 concentrations (0.5 ng/ml recommended) and exposure times

  • For immunostaining, use multiple markers (p-SMAD3, TGF-β, CTGF, and LMO7) to comprehensively assess pathway activity

  • When quantifying results, measure both the extent and intensity of staining/expression

• Translational implications:

  • LMO7's role suggests potential therapeutic targets for fibrotic diseases

  • Research methodologies should include assessment of whether interventions restore normal feedback regulation of TGF-β signaling

Understanding this negative feedback loop is crucial for interpreting experimental results in fibrosis research, as LMO7 deficiency may exacerbate TGF-β-driven fibrotic responses in multiple tissue contexts.

What techniques are recommended for studying LMO7's role in immune modulation?

To effectively investigate LMO7's emerging role in immune modulation, particularly its influence on regulatory T cells and immune evasion in cancer:

• Single-cell approaches:

  • Single-cell RNA sequencing is valuable for understanding the heterogeneity of immune microenvironments where LMO7 functions

  • This approach helped identify LMO7's association with Treg enrichment in pancreatic ductal adenocarcinoma

• Spatial analysis techniques:

  • Spatial proteomics and multiplex immunohistochemistry/immunofluorescence are recommended to visualize the relationship between LMO7-expressing tumor cells and infiltrating immune cells

  • These methods preserve tissue architecture while providing cellular resolution information

• Functional immunology assays:

  • T cell differentiation assays: To assess LMO7's effect on Treg differentiation

  • Chemotaxis assays: To evaluate LMO7's influence on immune cell recruitment

  • Cytotoxicity assays: To measure the impact on CD8+ T cell and NK cell function

• Mechanistic studies:

  • Ubiquitination assays to confirm LMO7's role in Foxp1 degradation

  • Chromatin immunoprecipitation to verify Foxp1 binding to TGF-β and CCL5 regulatory sites

  • Cytokine profiling to monitor TGF-β and CCL5 production in response to LMO7 manipulation

• In vivo models:

  • Syngeneic mouse models with LMO7 manipulation to study immune infiltration dynamics

  • Combine with immune checkpoint inhibitors to assess potential synergistic effects

  • Monitor survival outcomes alongside immune parameter changes

These methodological approaches should be integrated to develop a comprehensive understanding of how LMO7 regulates immune cell function, particularly in the context of cancer immunotherapy research.