ENKD1 Antibody

What is ENKD1 and why is it significant for cellular research?

ENKD1 is a protein of approximately 38.8 kDa (346 amino acids) that functions as a centrosomal component and microtubule-associated protein (MAP). Its significance lies in its role in ciliogenesis, particularly through competing with CEP97 for binding to CP110, which facilitates CP110 removal from the mother centriole - a critical step in initiating ciliogenesis . ENKD1 is expressed in multiple tissues, with particularly high expression in the testis and lung . Research has demonstrated that ENKD1 plays essential roles in regulating microtubule organization and stability, making it important for primary cilium formation and function .

Which detection methods work best for localizing ENKD1 in cellular compartments?

Immunofluorescence microscopy using specific antibodies against ENKD1 provides the most effective detection of ENKD1 at the centrosome and basal body of cilia. A recommended protocol includes:

• Fixation with 4% paraformaldehyde or methanol

• Permeabilization with 0.1-0.5% Triton X-100

• Blocking with BSA or normal serum

• Primary anti-ENKD1 antibody incubation (overnight at 4°C)

• Co-staining with centrosomal markers (e.g., Centrin) and ciliary markers (acetylated α-tubulin, Arl13b)

Super-resolution microscopy techniques provide superior resolution for precisely localizing ENKD1 within these structures . For confirming specificity, comparison of antibody reactivity between wild-type and ENKD1 knockout cells is essential, as demonstrated in studies using MEFs from ENKD1 knockout mice .

How can I validate ENKD1 antibody specificity for experimental applications?

Validating ENKD1 antibody specificity requires a multi-faceted approach:

• Genetic validation:

  • Compare antibody reactivity in wild-type versus ENKD1 knockout cells

  • Use siRNA-mediated knockdown samples as negative controls

• Western blot analysis:

  • Confirm single band detection at the expected 38.8 kDa size

  • Assess signal reduction in knockout/knockdown samples

• Immunofluorescence validation:

  • Verify centrosomal/basal body localization consistent with known ENKD1 distribution

  • Confirm signal disappearance in knockout/knockdown samples

• Overexpression controls:

  • Test antibody detection of tagged ENKD1 constructs

  • Compare endogenous versus overexpressed protein detection

Research has shown that proper validation should include comparing multiple tissues, as ENKD1 shows differential expression patterns, with notably high expression in bronchus, tonsil, adrenal gland, testis, and lung tissues .

What is the optimal experimental design to study ENKD1's role in ciliogenesis?

A comprehensive experimental design to study ENKD1's role in ciliogenesis should include:

• Depletion approaches:

  • siRNA-mediated knockdown in ciliated cell lines (RPE1, NIH3T3)

  • CRISPR/Cas9-mediated knockout in cells and animal models

  • Analysis of tissues from ENKD1 knockout mice

• Rescue experiments:

  • Re-expression of full-length ENKD1 in knockout/knockdown models

  • Domain-specific mutant expression (ENKD1-N, ENKD1-M, ENKD1-C) to identify functional regions

  • Quantitative assessment of rescue efficiency

• Mechanistic analysis:

  • Evaluation of CP110 and CEP97 localization at centrioles

  • Assessment of protein interactions through co-immunoprecipitation

  • Time-course experiments to monitor ciliogenesis progression

• Functional readouts:

  • Quantification of ciliated cell percentage

  • Measurement of ciliary length

  • Analysis of ciliary markers (acetylated α-tubulin, Arl13b)

  • Evaluation of ciliary signaling (e.g., Hedgehog pathway activation)

Studies have shown that the middle domain (91-250 aa) of ENKD1 is particularly important for ciliogenesis function, as it significantly rescues ENKD1 siRNA-induced ciliogenesis defects when re-expressed .

How should Western blot protocols be optimized for ENKD1 detection?

Optimizing Western blot protocols for ENKD1 detection requires attention to several key parameters:

• Sample preparation:

  • Use appropriate lysis buffers (RIPA or NP-40 based)

  • Include protease inhibitors to prevent degradation

  • For tissue samples, homogenization conditions should be optimized

• Protein separation:

  • Use 10-12% SDS-PAGE gels for optimal separation of the 38.8 kDa ENKD1 protein

  • Load 30-50 μg of total protein for adequate detection

  • Include molecular weight markers for accurate size determination

• Transfer and detection:

  • Semi-dry or wet transfer systems are suitable (100V/1 hour or 30V/overnight)

  • Block with 5% non-fat milk or BSA in TBST

  • Optimize primary antibody dilution (typically 1:500-1:2000)

  • Incubate with primary antibody overnight at 4°C

• Controls:

  • Include tissue samples with known high ENKD1 expression (testis, lung) as positive controls

  • Use ENKD1 knockout or knockdown samples as negative controls

  • Include loading controls (β-actin, GAPDH, or tubulin)

The specificity of the antibody should be validated by confirming a single band at approximately 38.8 kDa that disappears in knockout samples .

What methodologies are appropriate for investigating ENKD1's interaction with CP110 and CEP97?

To investigate ENKD1's competition with CEP97 for CP110 binding, implement these methodologies:

• Protein interaction assays:

  • Co-immunoprecipitation using antibodies against ENKD1, CP110, and CEP97

  • Proximity ligation assay (PLA) to visualize interactions in intact cells

  • Pull-down assays with recombinant proteins

  • Surface plasmon resonance (SPR) to measure binding kinetics

• Competition experiments:

  • Express CP110 with fixed amounts of tagged CEP97

  • Add increasing concentrations of ENKD1

  • Quantify changes in CEP97-CP110 complex formation

  • Perform reciprocal experiments with fixed ENKD1 and varying CEP97

• Domain mapping:

  • Generate truncation mutants of all three proteins

  • Identify minimal binding domains

  • Create point mutations in key residues to disrupt specific interactions

Research has demonstrated that ENKD1 depletion enhances the CP110-CEP97 interaction and detains CP110 at the mother centriole, suggesting competitive binding between ENKD1 and CEP97 . Time-course experiments have shown that with prolonged ENKD1 depletion (120h vs 48h), more cells display CP110 removal from the mother centriole, indicating the existence of inefficient ENKD1-independent mechanisms for CP110 removal .

How should ciliogenesis defects in ENKD1 knockout/knockdown models be quantified and analyzed?

For robust quantification and analysis of ciliogenesis defects in ENKD1 studies:

• Primary measurements:

  • Percentage of ciliated cells (minimum 300 cells per condition)

  • Ciliary length measurements (minimum 100 cilia per condition)

  • CP110 and CEP97 localization patterns (categorize as single or double dots)

• Quantification methodology:

  • Use random field selection to avoid bias

  • Employ automated image analysis when possible

  • Blind the experimenter to sample identity during quantification

• Statistical analysis:

  • Perform experiments in biological triplicates

  • Apply Student's t-test for comparing two conditions

  • Use ANOVA with appropriate post-hoc tests for multiple conditions

  • Present data as mean ± SD with individual data points shown

• Phenotypic categorization:

  Phenotype	Wild-type	ENKD1 KO	Statistical Significance
  Ciliated cells (%)	80.5 ± 5.2	32.3 ± 6.7	p < 0.001
  Ciliary length (μm)	3.8 ± 0.7	3.6 ± 0.8	p > 0.05 (NS)
  CP110 at both centrioles (%)	25.3 ± 4.2	72.8 ± 7.1	p < 0.001

Research has shown that ENKD1 knockout primarily affects cilia formation rather than ciliary length, with significant reductions in the percentage of ciliated cells in multiple tissues including retina, trachea, and cultured cells .

How can I interpret changes in CP110 localization after ENKD1 depletion?

When interpreting CP110 localization changes after ENKD1 depletion, consider these analytical frameworks:

• Quantitative assessment:

  • Calculate the percentage of cells with CP110 at both mother and daughter centrioles

  • Compare with control cells at multiple time points after serum starvation

  • Track temporal dynamics of CP110 removal

• Mechanistic interpretation:

  • Increased CP110 retention at the mother centriole suggests impaired removal

  • Enhanced CP110-CEP97 interaction indicates stabilization of the inhibitory complex

  • Time-dependent changes suggest alternative CP110 removal mechanisms

• Pattern analysis grid:

  Time point	CP110 pattern in control	CP110 pattern in ENKD1 KD	Interpretation
  24h starved	~30% double CP110 dots	~75% double CP110 dots	ENKD1 is required for efficient CP110 removal
  48h starved	~15% double CP110 dots	~65% double CP110 dots	Sustained defect in CP110 removal
  120h starved	~5% double CP110 dots	~35% double CP110 dots	Partial CP110 removal through alternative mechanisms

Research has demonstrated that upon ENKD1 depletion, the percentage of cells with strong CP110 and CEP97 signals at both mother and daughter centrioles significantly increases, confirming ENKD1's role in removing these proteins from the mother centriole . Interestingly, extended ENKD1 depletion (120h) shows improved CP110 removal compared to shorter depletion periods (48h), suggesting the existence of slower, ENKD1-independent CP110 removal mechanisms .

What approach should be used to analyze tissue-specific ENKD1 functions in knockout models?

To effectively analyze tissue-specific ENKD1 functions in knockout models:

• Comprehensive tissue analysis:

  • Examine multiple tissues with known ciliary functions

  • Use tissue-specific markers alongside ciliary markers

  • Compare tissues with different levels of ENKD1 expression

• Functional assessment methodologies:

  • Retina: Electroretinogram (ERG) and flash visual evoked potential (F-VEP)

  • Heart: Surface electrocardiography (ECG)

  • Sperm: Morphological analysis and motility assessment

  • Trachea: Scanning electron microscopy and immunostaining

• Quantitative comparison framework:

  Tissue	Parameter	Wild-type	ENKD1 KO	Functional Impact
  Retina	Photoreceptor cilia count	Normal	Decreased	Reduced ERG amplitude
  Heart	R-wave duration	Normal	Reduced	Altered cardiac conduction
  Trachea	Ciliated cells (%)	Normal	Decreased	Potential mucociliary clearance defects
  Testis	Sperm with tails (%)	Normal	Decreased	Retained fertility

Research on ENKD1 knockout mice has revealed tissue-specific phenotypes including reduced retinal photoreceptor cilia with consequent defective vision (demonstrated by reduced ERG amplitude), altered cardiac conduction, increased aberrant spermatozoa without tails, and reduced percentage of ciliated cells in the tracheal epithelium . Despite these defects, ENKD1 knockout mice show normal growth and survival when housed in pathogen-free conditions .

How can I distinguish between the functions of different ENKD1 domains in ciliogenesis?

To differentiate the functions of distinct ENKD1 domains in ciliogenesis:

• Domain-specific experimental approach:

  • Generate truncation mutants based on conserved domains (ENKD1-N: 1-91aa, ENKD1-M: 91-250aa, ENKD1-C: 250-346aa)

  • Create point mutations in conserved residues within each domain

  • Perform domain swapping with related proteins

• Rescue experiments:

  • Deplete endogenous ENKD1 using siRNA or CRISPR

  • Re-express domain-specific mutants

  • Quantify ciliogenesis restoration for each construct

• Domain function analysis matrix:

  ENKD1 Construct	Centrosomal Localization	CP110 Removal	Ciliogenesis Rescue	Microtubule Binding
  Full-length	+++	+++	+++	+++
  ENKD1-N (1-91)	+	+	+	+
  ENKD1-M (91-250)	+++	+++	+++	++
  ENKD1-C (250-346)	++	+	+	+++

Research has demonstrated that re-expression of full-length ENKD1 or the middle domain (ENKD1-M, 91-250aa) significantly rescues ENKD1 siRNA-induced ciliogenesis defects, while the N-terminal (ENKD1-N) and C-terminal (ENKD1-C) fragments do not . This indicates that the middle domain of ENKD1 is particularly critical for ciliogenesis function. The C-terminal fragment contains a conserved enkurin domain, which may have other functional roles .

What are the implications of ENKD1's role as a microtubule-associated protein for ciliary function?

The implications of ENKD1's role as a microtubule-associated protein for ciliary function are multifaceted:

• Microtubule dynamics regulation:

  • ENKD1 regulates microtubule organization and stability

  • Overexpression increases tubulin polymerization and microtubule stability

  • This affects the dynamic properties of the ciliary axoneme

• Ciliary structure maintenance:

  • As a MAP, ENKD1 may contribute to maintaining the 9+0 or 9+2 microtubule arrangement

  • It potentially stabilizes the ciliary axoneme through direct interaction with microtubules

  • It might regulate ciliary length by affecting microtubule dynamics

• Centriolar function:

  • ENKD1's centrosomal localization suggests roles in organizing centriolar microtubules

  • It facilitates CP110 removal, enabling the mother centriole to initiate axoneme extension

  • It may affect recruitment or organization of other centrosomal/basal body proteins

• Signaling implications:

  • Ciliary defects in ENKD1-depleted cells affect response to Hedgehog pathway activation

  • Proper microtubule organization is essential for ciliary trafficking and signaling

  • ENKD1 might facilitate the recruitment of signaling components to cilia

Research has shown that ENKD1 is not only localized to the centrosome but can also accumulate at the axoneme of cilia when overexpressed, and its overexpression induces the formation of microtubule bundles . This suggests that ENKD1 has direct effects on microtubule organization beyond its role in CP110 removal.

How can I design experiments to resolve contradictory findings in ENKD1 localization studies?

To resolve contradictions in ENKD1 localization studies, implement this systematic approach:

• Technical variables assessment:

  • Compare fixation methods (PFA vs. methanol vs. glutaraldehyde)

  • Test different permeabilization protocols (Triton X-100, saponin, digitonin)

  • Use multiple antibodies targeting different ENKD1 epitopes

  • Include appropriate controls (ENKD1 knockout samples)

• Complementary methodologies:

  • Compare immunofluorescence with live imaging of fluorescently tagged ENKD1

  • Use biochemical fractionation to isolate centrosomes and cilia

  • Employ super-resolution microscopy for higher precision localization

  • Validate with electron microscopy and immunogold labeling

• Systematic comparison grid:

  Technique	Fixative	Antibody/Tag	Observed Localization	Control Validation
  IF	PFA	Antibody A	Centrosome only	+/- ENKD1 KO
  IF	Methanol	Antibody B	Centrosome and cilia	+/- ENKD1 KO
  Live imaging	N/A	GFP-ENKD1	Centrosome ± cilia	Expression level check
  Super-resolution	PFA	Antibody C	Precise localization	+/- ENKD1 KO

Research has shown that ENKD1 maintains its centrosomal localization under both normal serum and serum-starved conditions . In ciliated cells, ENKD1 is detected at the basal body of the cilium. Interestingly, when overexpressed, ENKD1 localizes not only to the basal body but also accumulates at the axoneme of cilia, suggesting potential dosage-dependent localization patterns .

What approaches can be used to investigate ENKD1's role in ciliopathies and human disease?

To investigate ENKD1's potential role in ciliopathies and human disease:

• Genetic analysis approach:

  • Screen ENKD1 for variants in ciliopathy patient cohorts

  • Perform whole exome/genome sequencing in patients with unexplained ciliopathies

  • Analyze ENKD1 expression in ciliopathy-affected tissues

• Functional studies methodology:

  • Generate cellular models with patient-specific ENKD1 variants

  • Create animal models with equivalent mutations

  • Assess phenotypic similarities with human ciliopathy manifestations

• Clinical correlation assessment:

  • Compare ENKD1 knockout phenotypes with human ciliopathy features

  • Focus on retinal, renal, and neurological manifestations

  • Analyze expression in tissues commonly affected in ciliopathies

• Tissue-specific manifestation grid:

  Tissue	ENKD1 KO Phenotype	Related Human Ciliopathy	Potential Diagnostic Approach
  Retina	Reduced photoreceptor cilia	Retinitis pigmentosa	ERG analysis, retinal imaging
  Kidney	Not reported	Polycystic kidney disease	Renal function tests, ultrasound
  Brain	Not reported	Joubert syndrome	Neuroimaging, cognitive assessment
  Heart	Altered conduction	Heterotaxy, congenital heart defects	ECG, echocardiography

ENKD1 knockout mice display phenotypes in multiple organs including the retina (reduced photoreceptor cilia with consequent visual defects), heart (altered conduction system), and trachea (reduced percentage of ciliated cells) . These phenotypes show overlap with human ciliopathies, suggesting potential relevance to human disease. The fact that ENKD1 knockout mice exhibit significant but not lethal phenotypes suggests that mutations might contribute to milder forms of ciliopathies in humans.

What emerging technologies could advance our understanding of ENKD1 function?

Emerging technologies that could significantly advance ENKD1 research include:

• Advanced imaging approaches:

  • Super-resolution live imaging to track ENKD1 dynamics in real-time

  • Expansion microscopy for enhanced visualization of centrosomal/ciliary structures

  • Correlative light and electron microscopy (CLEM) for ultrastructural localization

  • Lattice light-sheet microscopy for long-term 3D imaging of ciliary processes

• Proximity labeling techniques:

  • BioID or APEX2 tagging of ENKD1 to identify proximal interactors

  • Domain-specific proximity labeling to map interaction landscapes

  • Temporal proximity labeling during ciliogenesis stages

• CRISPR-based technologies:

  • CRISPR activation/inhibition for controlled ENKD1 expression

  • Base editing for introducing specific mutations

  • CRISPR screens for identifying genetic interactions with ENKD1

• Structural biology approaches:

  • Cryo-EM of ENKD1-containing complexes

  • X-ray crystallography of ENKD1 domains with binding partners

  • Molecular dynamics simulations of ENKD1-microtubule interactions

These technologies would enable more precise characterization of ENKD1's molecular functions in ciliogenesis and microtubule regulation, potentially revealing new therapeutic targets for ciliopathies .

How can I integrate ENKD1 findings with broader ciliogenesis pathways?

To integrate ENKD1 findings with the broader ciliogenesis literature:

• Pathway mapping approach:

  • Place ENKD1 in established ciliogenesis pathways

  • Identify where ENKD1 function intersects with known mechanisms

  • Create comprehensive schematic models

• Comparative analysis methodology:

  • Compare ENKD1 knockout phenotypes with other ciliogenesis gene mutations

  • Identify similarities and differences in ciliary defects

  • Construct phenotypic clusters to identify functionally related genes

• Integrated pathway positioning:

  Ciliogenesis Step	Known Regulators	ENKD1's Role	Evidence	Research Gaps
  CP110 removal	CEP97, Talpid3, TTBK2	Competes with CEP97 for CP110 binding	Co-IP, rescue experiments	Upstream regulation
  Centrosome maturation	CEP164, CEP83, CEP89	Component of centrosome	Localization studies	Recruitment mechanisms
  Axoneme extension	IFT proteins, kinesins	Regulates microtubule stability	Overexpression effects	Direct vs. indirect effects
  Ciliary content regulation	Transition zone proteins	Required for proper content	KO phenotypes	Molecular mechanisms

Research has established that ENKD1 promotes CP110 removal through competing with CEP97, positioning it as a key regulator of an early step in ciliogenesis . Additionally, its role as a microtubule-associated protein places it among factors that regulate ciliary structure and stability . This dual function at both the initiation phase and structural maintenance phase of ciliogenesis makes ENKD1 an important integrator in ciliary biology.