DLEC1 Antibody

What is DLEC1 and what are its key characteristics relevant to antibody-based research?

DLEC1 (Deleted in Lung and Esophageal Cancer 1) is a tumor suppressor gene that is frequently downregulated in various human cancers. The protein contains multiple ASPM-SPD-2-Hydin (ASH) domains that facilitate protein-protein interactions and are commonly found in cilia/flagella or centrosome-associated proteins . DLEC1 is also known by several alternative names including F56, DLC1, DLC-1, and CFAP81 . The human DLEC1 protein is identified by Swiss-Prot accession number Q9Y238 with an NCBI Gene ID of 9940 . When working with DLEC1 antibodies, it's important to recognize that this protein has significant expression in testes, particularly in germ cells, which should be considered when designing experimental controls .

What are the standard specifications for DLEC1 antibodies used in research?

Commercial DLEC1 antibodies typically used in research are polyclonal antibodies raised in rabbits, purified through antigen affinity purification . These antibodies are typically designed to detect endogenous levels of total DLEC1 protein . The immunogen for these antibodies is often a synthetic peptide derived from human DLEC1 . For optimal experimental outcomes, DLEC1 antibodies are typically formulated in PBS (pH 7.4) with 0.05% NaN3 and 40% glycerol at concentrations of approximately 0.9mg/ml . Storage recommendations generally specify -20°C . The primary application validated for most commercial DLEC1 antibodies is immunohistochemistry (IHC), with demonstrated reactivity against human samples .

What considerations should be made regarding DLEC1 antibody specificity?

When selecting a DLEC1 antibody, researchers should verify that the antibody is capable of detecting endogenous levels of total DLEC1 protein rather than just overexpressed protein . The specificity should be validated through appropriate controls, especially since custom-made anti-DLEC1 antibodies may not always be suitable for all applications. For instance, research has shown that some anti-DLEC1 antibodies may not be appropriate for immunoprecipitation of endogenous DLEC1 . When studying mutant forms of DLEC1, such as truncated versions lacking specific domains (e.g., DLEC1ΔC that lacks the third ASH domain), researchers should verify antibody recognition of these variants, as expression levels may be significantly reduced (approximately one-tenth of wild-type DLEC1 expression) .

What are the optimal conditions for using DLEC1 antibodies in immunohistochemistry?

For effective immunohistochemistry experiments with DLEC1 antibodies, researchers should first perform antigen retrieval to ensure optimal epitope exposure. The antibody should be diluted to working concentration (typically 1:100 to 1:500, but optimization is required) in a blocking buffer containing 1-5% BSA or normal serum from the same species as the secondary antibody. Incubation should be performed overnight at 4°C to maximize specific binding . When designing these experiments, it's important to note that DLEC1 shows variable expression across tissues, with particularly strong expression in testes . Therefore, appropriate positive and negative control tissues should be included. Validation studies have confirmed that commercially available DLEC1 antibodies can detect endogenous levels of total DLEC1 protein in human samples , but optimization may be required for specific tissue types.

How can researchers effectively validate DLEC1 knockout or knockdown models?

Validation of DLEC1 knockout models requires a multi-faceted approach. Genomic verification through PCR is essential to confirm deletion of the targeted exons, as demonstrated in studies using CRISPR-Cas9 to delete exons 28-33 of DLEC1 . Researchers should also perform RT-PCR to detect potential truncated mRNA transcripts that might still be expressed despite genomic deletion . Western blotting is crucial to confirm the absence of the full-length protein and to detect any potential truncated protein products that might retain partial functionality. In one study, a truncated form of DLEC1 (DLEC1ΔC) was detected at approximately one-tenth the expression level of wild-type DLEC1 . Functional assays relevant to the known roles of DLEC1 should be performed to confirm phenotypic changes. These may include assessing spermatogenesis in male mice or evaluating ciliogenesis in cell culture models . Additionally, researchers should verify the absence of off-target effects by sequencing potential off-target sites for each guide RNA used in CRISPR-Cas9 approaches .

What protocols are recommended for investigating DLEC1 protein-protein interactions?

For studying DLEC1 protein-protein interactions, immunoprecipitation followed by mass spectrometry has proven effective. Since commercial anti-DLEC1 antibodies may not be optimal for immunoprecipitation of endogenous DLEC1, expressing tagged versions (e.g., 3×FLAG-tagged human DLEC1) in appropriate cell lines such as HEK293F cells is recommended . The protocol involves:

• Transfecting cells with 3×FLAG-tagged DLEC1 or empty vector control

• Lysing cells with 1% Triton X-100 buffer

• Removing insoluble material through centrifugation

• Incubating approximately 20mg of lysate with 40μL of anti-FLAG antibody-conjugated agarose beads overnight at 4°C

• Washing the immunoprecipitants thoroughly

• Eluting bound proteins with 2-mercaptoethanol-free sample buffer at 37°C for 30 minutes

• Analyzing co-immunoprecipitated proteins by western blotting or liquid chromatography-tandem mass spectrometry (LC-MS/MS)

This approach has successfully identified interactions between DLEC1 and numerous proteins including α- and β-tubulin, TRiC subunits, and Bardet-Biedl Syndrome (BBS) protein complex components .

What is currently known about DLEC1's molecular interactions and their functional significance?

DLEC1 has been found to interact with several important protein complexes that provide insight into its molecular function. Mass spectrometry and co-immunoprecipitation studies have identified 67 DLEC1-interacting proteins, many of which are associated with cilia and flagella formation . Key interactions include:

• Tubulins: DLEC1 interacts with α- and β-tubulin, the main structural components of axonemes in cilia and flagella, but shows limited binding to γ-tubulin .

• TRiC Complex: DLEC1 binds to multiple T-complex protein 1 (TCP-1) family proteins that form the tailless complex polypeptide 1 ring complex (TRiC), which functions as a chaperone for tubulin and actin, enhances retrograde axonal transport, and mediates BBSome assembly .

• BBS Proteins: DLEC1 interacts with several Bardet-Biedl Syndrome (BBS) proteins including BBS2, BBS4, BBS5, and BBS6, which are involved in the recognition and trafficking of cargo in or to cilia and flagella .

These interactions suggest DLEC1 plays a critical role in intraflagellar transport and ciliogenesis, particularly in the formation of sperm flagella. The protein appears to regulate protein transport from cytoplasm to primary cilia rather than functioning within the cilia themselves .

How does DLEC1 contribute to spermatogenesis and what insights does this provide for research applications?

DLEC1 plays an essential role in spermatogenesis, particularly during the elongation phase of spermatid development. Research using DLEC1-knockout mice has revealed that:

• Spermatogenesis progresses normally to step 8 spermatids in DLEC1-deficient mice but exhibits abnormalities during the elongation phase .

• DLEC1-deficient elongating spermatids display head deformation, shortened tails, and abnormal manchette organization .

• These phenotypes closely resemble those observed in mice deficient in various intraflagellar transport (IFT)-associated genes .

• Despite the formation of the manchette structure in DLEC1-deficient spermatids, proper organization is disrupted .

These findings suggest DLEC1 functions as a regulator of intraflagellar transport, playing a crucial role in sperm head and tail formation. When designing experiments to study DLEC1 function, researchers should focus on cellular models that require functional IFT or cilia formation. The increased resistance of DLEC1 to Triton X-100 extraction in mature sperm compared to immature germ cells suggests the protein is transported from cytosol to flagella during spermatogenesis, a feature that can be utilized in fractionation studies .

What experimental approaches can be used to study DLEC1's role in ciliogenesis?

To investigate DLEC1's role in ciliogenesis, several experimental approaches have proven effective:

• Stable expression systems: Establishing cell lines stably expressing human DLEC1 (hDLEC1) in appropriate models such as A549 lung adenocarcinoma cells, which undergo ciliogenesis upon serum starvation .

• Cilia detection and quantification: Immunostaining with cilia markers such as anti-ARL13B antibody, followed by quantitative analysis of:

  • Percentage of ciliated cells

  • Ciliary length

  • Morphological characteristics

• Tubulin polymerization assays: Assessing whether DLEC1 expression affects tubulin polymerization in cell-based systems or in tissue samples from wild-type versus DLEC1-knockout animals .

• Subcellular localization studies: Investigating the intracellular distribution of DLEC1 through immunofluorescence or by utilizing tagged versions of the protein .

Research has shown that DLEC1 expression in A549 cells increases both the percentage of ciliated cells and ciliary length, suggesting it enhances ciliogenesis . When conducting such experiments, it's important to note that DLEC1 appears to be relatively uniformly distributed in the cytoplasm or as indistinct dot-like structures rather than localizing to primary cilia themselves .

How should researchers analyze protein interaction data involving DLEC1?

When analyzing DLEC1 protein interaction data, researchers should:

• Prioritize functional grouping: Organize identified interacting proteins based on their known functions and cellular localizations. As shown in previous studies, DLEC1 interactors can be grouped into categories such as axonemal structural components, chaperone proteins, and IFT-associated factors .

• Validate key interactions: Confirm interactions of interest through orthogonal methods. For example, mass spectrometry findings should be validated through co-immunoprecipitation followed by western blotting, as was done to confirm DLEC1's interaction with α- and β-tubulin, TRiC subunits, and BBS proteins .

• Consider interaction significance: Evaluate interaction strength using quantitative metrics such as the "−10lg P" scores reported in LC-MS/MS data, as shown in the table below from previous DLEC1 interaction studies:

• Look beyond direct identifications: Consider functional partners that may not be directly identified but are part of the same complexes. For instance, although mass spectrometry didn't detect BBSome subunits directly, subsequent testing revealed DLEC1's interaction with multiple BBS proteins .

What control experiments are essential when studying DLEC1 using antibody-based techniques?

When employing antibody-based techniques to study DLEC1, several essential control experiments should be included:

• Antibody validation controls:

  • Utilize DLEC1 knockout or knockdown samples to confirm antibody specificity

  • Test antibody recognition across multiple species if working with non-human models

  • Verify antibody performance in all intended applications (IHC, western blotting, etc.)

• Immunohistochemistry controls:

  • Include positive control tissues known to express DLEC1 (such as testes)

  • Incorporate negative control tissues or DLEC1-knockout samples

  • Perform secondary antibody-only controls to assess non-specific binding

  • Use isotype controls to evaluate potential background

• Protein interaction controls:

  • When performing co-immunoprecipitation, include an empty vector or non-relevant protein control

  • For FLAG-tagged DLEC1 pull-downs, incorporate FLAG peptide competition controls

  • Test reciprocal immunoprecipitation when possible (pull down interacting protein and probe for DLEC1)

• Subcellular fractionation controls:

  • Include markers for different cellular compartments (cytosolic, nuclear, membrane-bound)

  • Compare fractionation patterns between wild-type and DLEC1-deficient samples

  • Consider resistance to detergent extraction as a measure of protein association with structural elements (as seen with DLEC1's Triton X-100 resistance in mature sperm)

How can researchers reconcile DLEC1's dual roles as a tumor suppressor and regulator of ciliogenesis?

Analyzing DLEC1's apparently distinct roles requires careful experimental design and integrative data interpretation:

• Mechanistic overlap investigation: Researchers should design experiments to determine whether there are shared molecular mechanisms between DLEC1's tumor suppressor function and its role in ciliogenesis. For instance, examining whether DLEC1's interactions with the TRiC complex or tubulin affect both cell cycle regulation and cilia formation.

• Cell type-specific function analysis: Compare DLEC1's molecular interactions and effects across different cell types, particularly contrasting cancer cells with highly ciliated cells like spermatocytes. Research has shown that while DLEC1 is downregulated in various cancers through promoter hypermethylation , it is highly expressed in testes, particularly in germ cells .

• Domain-function correlation: Utilize truncation or point mutation studies to identify which domains of DLEC1 are essential for its different functions. The presence of multiple ASH domains, which are often found in cilia/flagella or centrosome-associated proteins , suggests functional specialization within the protein.

• Signaling pathway integration: Investigate how DLEC1 might integrate into signaling pathways relevant to both cancer development and ciliogenesis, such as Hedgehog or Wnt signaling, which rely on primary cilia for proper function.

• Expression restoration experiments: In cancer cells with hypermethylated DLEC1 promoters, conduct complementary experiments using 5-aza-2′-deoxycytidine to induce DNA demethylation alongside ciliogenesis assays to determine if restoration of DLEC1 expression simultaneously inhibits cell proliferation and enhances ciliogenesis .

What experimental strategies can identify the role of DLEC1 in intraflagellar transport?

To elucidate DLEC1's role in intraflagellar transport (IFT), researchers should consider these advanced experimental approaches:

• Live cell imaging of IFT: Utilize fluorescently tagged IFT proteins in combination with DLEC1 expression or knockout to visualize IFT dynamics in real-time. This approach can reveal whether DLEC1 affects the speed, frequency, or directionality of IFT particle movement.

• Biochemical fractionation of IFT complexes: Isolate IFT-A and IFT-B complexes from cells with and without DLEC1 to determine if DLEC1 affects complex composition or stability. Given DLEC1's interaction with BBSome components , particular attention should be paid to cargo recognition and loading.

• DLEC1 mutational analysis: Generate mutations in specific ASH domains of DLEC1 to determine which regions are crucial for interaction with IFT components. Since DLEC1 contains multiple ASH domains commonly found in cilia/flagella-associated proteins , domain-specific analysis can reveal functional specialization.

• Comparative analysis with established IFT mutants: Compare the phenotypic effects of DLEC1 deficiency with those of known IFT component mutations. The similarity of DLEC1-knockout mouse spermatid abnormalities to those seen in IFT-associated gene deficiencies provides a foundation for such comparisons .

• Cargo tracking assays: Use fluorescently tagged cargoes known to be transported by IFT (such as tubulin) to determine if DLEC1 affects cargo selection, loading, or delivery. DLEC1's enhancement of ciliogenesis in A549 cells suggests it may facilitate protein transport to primary cilia .

How can researchers effectively study the relationship between DLEC1 and the TRiC/CCT chaperonin complex?

To investigate the functional relationship between DLEC1 and the TRiC/CCT chaperonin complex, researchers should implement these specialized approaches:

• In vitro chaperonin activity assays: Assess whether DLEC1 affects the chaperone activity of TRiC/CCT complexes using purified components and model substrates such as tubulin and actin.

• TRiC/CCT subunit depletion studies: Systematically deplete individual TRiC/CCT subunits and evaluate the impact on DLEC1's ability to enhance ciliogenesis or regulate spermatogenesis. DLEC1 has been shown to interact with multiple TRiC/CCT subunits including TCP-1 α, TCP-1 β, TCP-1 γ, TCP-1 δ, TCP-1 ε, and TCP-1 η .

• BBSome assembly analysis: Examine whether DLEC1 influences the TRiC/CCT-mediated assembly of the BBSome complex, which is critical for ciliary trafficking. DLEC1 interacts with both TRiC/CCT components and multiple BBS proteins (BBS2, BBS4, BBS5, and BBS6) .

• Domain mapping of interactions: Determine which domains of DLEC1 and TRiC/CCT subunits are responsible for their interaction through truncation and point mutation studies.

• Structural biology approaches: Employ cryo-electron microscopy or X-ray crystallography to characterize the structural basis of DLEC1-TRiC/CCT interactions, which could reveal how DLEC1 might influence chaperonin function.

• Subcellular co-localization studies: Investigate where in the cell DLEC1 and TRiC/CCT components interact, paying particular attention to the centrosome and basal body regions that are critical for ciliogenesis.

What approaches can resolve contradictory data regarding DLEC1 localization in cilia and flagella?

To address conflicting findings regarding DLEC1's localization in cilia and flagella, researchers should employ multiple complementary approaches:

• Improved immunodetection methods: Develop higher-sensitivity antibodies or amplification techniques for detecting endogenous DLEC1, as previous studies noted difficulties in obtaining antibodies suitable for immunostaining or detecting low expression levels of tagged DLEC1 in knock-in mice .

• Alternative tagging strategies: Test different epitope tags (beyond FLAG) and various insertion positions to minimize disruption of DLEC1 localization signals. Consider smaller tags or split complementation approaches that might be less disruptive to protein targeting.

• Super-resolution microscopy: Employ techniques such as STORM, PALM, or SIM to achieve nanoscale resolution of DLEC1 localization relative to known ciliary markers and subdomains.

• Comparative analysis across species: Study DLEC1 localization across multiple model organisms, particularly focusing on Chlamydomonas reinhardtii, where the DLEC1 ortholog FAP81 has been identified as a flagellar protein .

• Cell-type specific analysis: Compare DLEC1 localization across different ciliated cell types, noting that localization patterns in primary cilia (which lack central pairs of microtubules) may differ from those in motile cilia or sperm flagella .

• Biochemical fractionation with graduated detergent extraction: Perform sequential extractions with increasing detergent concentrations to determine DLEC1's association with different ciliary compartments, building on observations that DLEC1 in sperm is more resistant to Triton X-100 extraction compared to DLEC1 in testes containing immature germ cells .

What are the most promising future research directions for DLEC1 antibody applications?

Future research using DLEC1 antibodies should focus on several key areas:

• Development of phospho-specific antibodies: Creating antibodies that recognize specific post-translational modifications of DLEC1 could reveal regulatory mechanisms controlling its function in different cellular contexts.

• Domain-specific antibodies: Generating antibodies targeting individual ASH domains could help dissect the functional roles of different regions of DLEC1 in protein-protein interactions.

• Cross-species comparative studies: Developing antibodies with verified cross-reactivity across model organisms would facilitate evolutionary and comparative studies of DLEC1 function, particularly between humans and model organisms like mice.

• Antibody-based proximity labeling: Implementing BioID or APEX2 fusion approaches with DLEC1 could map its proximal interactome in living cells, potentially revealing transient or context-specific interactions missed by traditional co-immunoprecipitation approaches.

• Single-cell analysis applications: Optimizing DLEC1 antibodies for techniques like imaging mass cytometry or cyclic immunofluorescence could reveal cell-to-cell variability in DLEC1 expression and function within heterogeneous tissues.

• Therapeutic targeting approaches: Investigating whether DLEC1 antibodies could be used to modulate its function in disease contexts, particularly in cancers where DLEC1 is downregulated.