LZTFL1 Antibody

What is LZTFL1 and why is it important for research?

LZTFL1 is a 299 amino acid protein with a molecular weight of approximately 35 kDa that contains a basic region, a coil-coil domain, and a leucine zipper domain . It was initially identified through elimination testing and subsequent genomic sequencing and cDNA cloning . LZTFL1 has gained significant research interest due to its:

• Role as a tumor suppressor in various cancers, with expression significantly downregulated in tumor samples compared to normal tissues

• Association with patient survival outcomes and inverse correlation with tumor metastasis

• Involvement in epithelial cell differentiation with expression patterns that overlap with E-cadherin at the plasma membrane

• Identification as a candidate effector gene at a COVID-19 risk locus, with strong expression in lung tissue and particularly in ciliated cells of the respiratory epithelium

The multifaceted functions of LZTFL1 make it an important target for research in oncology, respiratory diseases, and developmental biology.

What are the typical applications for LZTFL1 antibodies in research?

LZTFL1 antibodies can be employed in various experimental techniques, including:

• Western Blot (WB): For detecting LZTFL1 protein expression in cell and tissue lysates, with recommended dilutions ranging from 1:5000 to 1:50000

• Immunofluorescence (IF)/Immunocytochemistry (ICC): For visualizing subcellular localization of LZTFL1, with recommended dilutions of 1:400 to 1:1600

• Flow Cytometry (FC): For detecting intracellular LZTFL1 expression at a concentration of 0.40 μg per 10^6 cells in a 100 μl suspension

• Immunohistochemistry (IHC): For examining LZTFL1 expression in tissue sections, as demonstrated in studies of normal and cancerous tissues

• Co-immunoprecipitation: For investigating protein-protein interactions involving LZTFL1

These applications allow researchers to investigate LZTFL1 expression, localization, and interactions in various experimental contexts.

What are the common models and samples used to study LZTFL1?

Based on the research literature, LZTFL1 has been studied in:

• Cell lines: Multiple human cell lines including HCT 116, LNCaP, HEK-293, HeLa, Jurkat, and K-562 cells have been used to study LZTFL1 expression and function

• Tissue samples: LZTFL1 expression has been examined in various normal human tissues including breast, esophagus, pancreas, stomach, ovary, prostate, lung, colon, thyroid, kidney, bladder, and liver, as well as their corresponding cancer samples

• Mouse models: Studies have investigated LZTFL1 expression in multiple mouse tissues including heart, brain, spleen, lung, liver, kidney, muscle, and testis, with particularly high expression observed in testicular tissue

• Primary cells: Single-cell RNA-seq has shown LZTFL1 expression throughout the respiratory epithelium, predominantly in ciliated cells

This diversity of models allows for comprehensive investigation of LZTFL1 functions across different biological contexts.

How can researchers optimize Western blot protocols for LZTFL1 detection?

Optimizing Western blot protocols for LZTFL1 detection requires careful consideration of several factors:

• Protein extraction: Based on published protocols, extraction buffers containing 50 mM Tris–HCl pH 8.0, 170 mM NaCl, 1% NP40, 5 mM EDTA, 1 mM DTT, and protease inhibitors have been successfully used for LZTFL1 detection

• Protein loading: Detection sensitivity can vary significantly based on loading amount. Studies have shown that while 50 μg of total protein may be sufficient for detecting LZTFL1 in tissues with high expression (e.g., testis), up to 100 μg may be needed for tissues with lower expression levels

• Antibody concentration: Antibody dilutions can be adjusted based on expression levels, with ranges from 1:400 for low-expressing tissues to 1:2000 for high-expressing tissues

• Detection system: The choice between standard (e.g., Pico) and high-sensitivity (e.g., Femto) chemiluminescence systems can significantly impact detection, especially for tissues with low LZTFL1 expression

• Molecular weight confirmation: Always confirm LZTFL1 detection at the expected molecular weight of approximately 35 kDa

These considerations are crucial for obtaining reliable and reproducible results when detecting LZTFL1 by Western blot.

What methods are recommended for studying LZTFL1 expression in specific cell types within a tissue?

For investigating cell type-specific expression of LZTFL1, several complementary approaches are recommended:

• Single-cell RNA sequencing (scRNA-seq): This approach has successfully identified LZTFL1 expression patterns across different cell types in the respiratory epithelium, showing predominant expression in ciliated cells

• Immunohistochemistry with detailed scoring: Studies have employed tissue microarrays and clinical specimens with systematic scoring methods to assess LZTFL1 expression levels across different cell types and in normal versus cancerous samples

• Dual immunofluorescence staining: Co-staining with LZTFL1 antibody and cell type-specific markers (e.g., α-tubulin) allows for precise localization of LZTFL1 in specific cell populations

• Isolation of specific cell populations: For tissues like testis, protocols for isolating specific cell populations (e.g., spermatogenic cells) followed by immunofluorescence have been effective for studying LZTFL1 expression during differentiation processes

• Temporal expression analysis: Examining LZTFL1 expression during developmental processes (e.g., first wave of spermatogenesis) can provide insights into its role in cell differentiation

These methods provide complementary information about LZTFL1 expression patterns at cellular and subcellular levels.

How can researchers investigate the functional roles of LZTFL1 in cellular processes?

To elucidate the functional roles of LZTFL1 in cellular processes, researchers have employed several experimental strategies:

• Gene overexpression: Construction of expression vectors (e.g., pcDNA-Flag-LZTFL1, pTRE2-LZTFL1-ires-EGFP) for transfection into cell lines has been used to study the effects of LZTFL1 overexpression on cell behavior

• Inducible expression systems: Doxycycline-inducible systems (e.g., Hela-tet-on cells with pTRE2-LZTFL1-ires-EGFP) allow for controlled expression of LZTFL1 to study its effects on cellular phenotypes

• RNA interference: LZTFL1-specific siRNA duplexes have been used to knock down LZTFL1 expression and assess resulting changes in cellular functions

• Protein-protein interaction studies: Co-immunoprecipitation assays have been employed to identify interaction partners of LZTFL1, providing insights into its molecular functions

• In vivo models: Xenograft models have been used to assess the effects of LZTFL1 expression on tumor growth

These approaches provide complementary information about LZTFL1's roles in cellular processes such as proliferation, migration, and differentiation.

What are the main challenges in detecting endogenous LZTFL1 in different tissue types?

Several challenges exist when detecting endogenous LZTFL1 across different tissue types:

• Varying expression levels: LZTFL1 expression shows significant tissue-specific variation. While it is highly expressed in testis, its expression in other tissues like brain, spleen, lung, and liver may require more sensitive detection methods

• Antibody sensitivity and specificity: The quality of the antibody is crucial for reliable detection. Researchers should validate antibodies using positive controls with known LZTFL1 expression and negative controls (e.g., LZTFL1 knockdown samples)

• Detection system sensitivity: Different tissues may require different detection sensitivities. For example, studies have shown that while the Pico system may be sufficient for high-expressing tissues, the more sensitive Femto system may be needed for tissues with lower expression

• Sample preparation: Proper tissue fixation and processing are critical, especially for immunohistochemistry. Overfixation can mask epitopes, while inadequate fixation may affect tissue morphology

• Background signal: Non-specific binding can complicate interpretation, particularly in tissues with complex cellular composition. Proper blocking and antibody dilution optimization are essential

Addressing these challenges requires careful technique optimization and the use of appropriate controls.

How can researchers validate the specificity of LZTFL1 antibodies in their experimental systems?

Validation of LZTFL1 antibody specificity is critical for ensuring reliable experimental results. Recommended validation approaches include:

• Multiple antibody comparison: Testing different LZTFL1 antibodies (e.g., commercially available ones and lab-generated ones) to confirm consistent detection patterns

• Positive and negative controls: Using cell lines or tissues with known high expression of LZTFL1 (e.g., testis, epithelial cells) as positive controls and appropriate negative controls

• LZTFL1 knockdown or knockout: Confirming reduced or absent antibody signal in samples where LZTFL1 has been knocked down using siRNA or knocked out using CRISPR-Cas9

• LZTFL1 overexpression: Verifying increased signal in samples where LZTFL1 has been overexpressed (e.g., cells transfected with LZTFL1 expression vectors)

• Western blot molecular weight confirmation: Ensuring that the detected band corresponds to the expected molecular weight of LZTFL1 (approximately 35 kDa)

• Peptide competition assay: Pre-incubating the antibody with the immunizing peptide should abolish specific staining if the antibody is specific

These validation steps help ensure that experimental observations accurately reflect LZTFL1 biology rather than artifacts or non-specific interactions.

How should researchers quantify and analyze LZTFL1 expression data in clinical samples?

For robust quantification and analysis of LZTFL1 expression in clinical samples, researchers should consider the following approaches:

• Immunohistochemistry scoring systems: Studies have successfully employed scoring systems based on staining intensity and percentage of positive cells. For example, a 0-9 scoring system was used in gastric cancer studies, with scores ≤4 and >4 used for statistical analyses

• Correlation with clinical parameters: Statistical analyses such as Pearson correlation can be used to assess relationships between LZTFL1 expression levels and clinical features (e.g., age, sex, tumor stage, metastasis)

• Survival analysis: Kaplan-Meier survival curves and log-rank tests can be employed to determine whether LZTFL1 expression correlates with patient outcomes

• Multiple tissue comparison: When analyzing LZTFL1 expression across different tissues, normalization to appropriate housekeeping genes or proteins (e.g., GAPDH) is essential

• Integration with genomic data: For comprehensive analysis, LZTFL1 expression data can be integrated with genetic information, such as the presence of specific alleles (e.g., rs17713054) that may affect expression levels

This table demonstrates correlation analysis between LZTFL1 expression and clinical parameters in gastric cancer patients .

What are the important considerations when analyzing LZTFL1 localization data?

When analyzing LZTFL1 localization data, researchers should consider several factors:

• Subcellular compartmentalization: LZTFL1 has been observed at the plasma membrane, overlapping with E-cadherin expression, suggesting potential roles in cell-cell adhesion . Careful co-localization studies with markers for different cellular compartments are important for accurate interpretation.

• Cell type-specific localization: LZTFL1 shows differential expression across cell types within tissues. For example, in respiratory epithelium, it is predominantly expressed in ciliated cells . Cell type identification should be confirmed with appropriate markers.

• Temporal changes in localization: LZTFL1 expression and localization can change during differentiation processes, as observed during spermatogenesis . Time-course analyses may be necessary to capture such dynamics.

• Resolution limitations: Standard immunofluorescence may not provide sufficient resolution for precise localization. Super-resolution microscopy techniques may be required for detailed subcellular localization studies.

• Fixation and permeabilization effects: Different fixation methods can affect antigen preservation and accessibility. Comparison of multiple fixation protocols (e.g., PFA, methanol) may be necessary for comprehensive localization analysis .

These considerations help ensure accurate interpretation of LZTFL1 localization data and its biological implications.

How can LZTFL1 antibodies be utilized to study its role in cancer progression?

LZTFL1 antibodies can be employed in multiple ways to investigate its role in cancer progression:

• Expression profiling across cancer types: Immunohistochemical analysis using tissue microarrays has revealed that LZTFL1 is significantly downregulated in multiple cancer types compared to normal tissues, suggesting a tumor suppressor role

• Correlation with clinicopathological features: Studies using LZTFL1 antibodies have demonstrated significant inverse correlation between LZTFL1 expression and tumor metastasis, as well as positive correlation with patient survival in gastric cancer

• Cell-based functional assays: LZTFL1 antibodies can be used to confirm the expression of LZTFL1 in cells transfected with LZTFL1 expression vectors to study its effects on cancer-related phenotypes such as anchorage-independent growth and cell migration

• Studying molecular mechanisms: Co-immunoprecipitation using LZTFL1 antibodies can identify interaction partners that may mediate its tumor suppressive functions

• Epithelial-mesenchymal transition (EMT) studies: Given LZTFL1's association with epithelial differentiation and its co-localization with E-cadherin, antibodies can be used to investigate its role in EMT, a process central to cancer progression and metastasis

These approaches provide complementary insights into how LZTFL1 functions in cancer biology and its potential as a biomarker or therapeutic target.

What are the emerging research areas where LZTFL1 antibodies may be particularly valuable?

Several emerging research areas highlight the increasing value of LZTFL1 antibodies:

• COVID-19 pathogenesis: LZTFL1 has been identified as a candidate effector gene at a COVID-19 risk locus, with the rs17713054 A risk allele associated with higher LZTFL1 expression in the lung . Antibodies can help elucidate how LZTFL1 contributes to COVID-19 susceptibility and severity.

• Ciliopathies and ciliary function: Given LZTFL1's expression in ciliated cells and its classification as an intraflagellar protein, antibodies can facilitate studies of its role in ciliary functions and ciliopathies .

• Epithelial-mesenchymal transition in development and disease: The association of LZTFL1 with epithelial differentiation suggests important roles in EMT. Antibodies can help map its expression patterns during normal development and disease-related EMT processes .

• Spermatogenesis and male fertility: The high expression of LZTFL1 in testis and its dynamic regulation during spermatogenesis point to potential roles in male reproductive functions that can be investigated using LZTFL1 antibodies .

• Biomarker development: The correlation between LZTFL1 expression and clinical outcomes in cancer patients suggests potential as a prognostic biomarker, which could be assessed in larger cohorts using antibody-based techniques .

These research directions represent promising areas where LZTFL1 antibodies can contribute to significant scientific and clinical advances.