DCTN4 Antibody

What is DCTN4 and what cellular functions does it mediate?

DCTN4 (Dynactin subunit 4, also known as p62) is an integral component of the dynactin multisubunit complex, which serves as a required cofactor for most cellular processes powered by the microtubule-based motor cytoplasmic dynein . DCTN4 contains a highly conserved cysteine-rich motif that interacts directly with Arp1 (actin-related protein 1) . Functionally, DCTN4 has a dual role in dynein targeting and in Arp1 subunit pointed-end capping within the dynactin complex . It is implicated in linking dynein and dynactin to the cortical cytoskeleton . As part of the pointed-end complex of the dynactin shoulder, DCTN4 associates with DCTN5, DCTN6, and ACTR10 subunits, with direct binding to the ACTR1A subunit .

What is the subcellular localization pattern of DCTN4?

DCTN4 exhibits a distinctive subcellular distribution pattern that reflects its functional roles. It displays a punctate cytoplasmic distribution as well as centrosomal localization that is characteristic of dynactin components . Interestingly, while overexpression of DCTN4 does not disrupt microtubule organization or compromise the integrity of the Golgi apparatus, it can result in both cytosolic and nuclear distribution . This observation suggests that at very high expression levels, DCTN4 may be targeted to the nucleus, potentially indicating additional functions beyond its canonical cytoskeletal roles .

What types of DCTN4 antibodies are available for research applications?

The primary types of DCTN4 antibodies available for research include polyclonal antibodies, such as the rabbit polyclonal antibody described in the search results . These antibodies are typically generated by immunizing host animals (usually rabbits) with recombinant human DCTN4 protein . The polyclonal nature of these antibodies means they recognize multiple epitopes on the DCTN4 protein, which can enhance detection sensitivity in various applications. These antibodies may show cross-reactivity across species, with many commercial DCTN4 antibodies demonstrating reactivity with human, mouse, and rat samples .

What is the clinical significance of DCTN4 in human disease?

DCTN4 has been identified through exome sequencing as a modifier gene associated with Pseudomonas aeruginosa infection susceptibility in cystic fibrosis patients . Specific missense variants in DCTN4 (rs11954652; Phe349Leu and rs35772018; Tyr270Cys) are associated with earlier age of first P. aeruginosa infection, faster progression to chronic infection, and earlier conversion to mucoid P. aeruginosa . This association is particularly significant because DCTN4 functions as part of the dynein-dependent motor that moves autophagosomes along microtubules into lysosomes for degradation during autophagy—a critical cellular quality control mechanism for transporting and degrading damaged proteins and microbes . This finding represents one of the first discoveries of a gene for a complex trait using exome sequencing of extreme phenotypes .

How can DCTN4 antibodies be effectively used in immunohistochemistry (IHC) applications?

For optimal IHC applications with DCTN4 antibodies, researchers should consider a dilution range of 1:100-1:300 as recommended for commercial polyclonal antibodies . The antibody has been verified for use with human ovarian cancer and human cervical cancer samples . When designing IHC experiments, consider that DCTN4 has a punctate cytoplasmic and centrosomal distribution pattern, which should guide your evaluation of staining results .

For IHC protocol optimization:

• Use appropriate antigen retrieval methods (typically heat-induced epitope retrieval in citrate buffer pH 6.0)

• Incorporate proper blocking steps to minimize background

• Incubate with the primary antibody overnight at 4°C for best results

• Include positive controls (verified samples such as ovarian or cervical cancer tissues) and negative controls (omitting primary antibody)

• Evaluate staining patterns with attention to both cytoplasmic punctate and centrosomal localization

What experimental approaches can be used to study DCTN4's role in autophagy and host defense against pathogens?

Given DCTN4's involvement in the dynein-dependent motor that facilitates autophagosome transport during autophagy and its association with susceptibility to P. aeruginosa infection in cystic fibrosis , several experimental approaches can be employed:

• Autophagy flux assays: Monitor LC3-II levels in the presence/absence of DCTN4 knockdown or overexpression to assess autophagosome formation and clearance

• Live cell imaging: Visualize autophagosome trafficking along microtubules in cells expressing fluorescently-tagged DCTN4 and autophagy markers

• Infection models: Establish cellular or animal models with DCTN4 variants to study pathogen clearance efficiency

• Co-immunoprecipitation: Investigate DCTN4's interaction with other components of the dynactin complex and autophagy machinery

• CRISPR/Cas9 genome editing: Generate cell lines harboring DCTN4 variants identified in cystic fibrosis patients (such as rs11954652 and rs35772018) to study functional consequences

These approaches can help elucidate how DCTN4 variants might compromise autophagy-dependent pathogen clearance mechanisms, potentially explaining the increased susceptibility to P. aeruginosa infections observed in carriers of specific DCTN4 variants.

How can researchers investigate the interaction between DCTN4 and the Arp1 filament in the dynactin complex?

To study the interaction between DCTN4 and Arp1 (ACTR1A) within the dynactin complex:

• Structural analysis: Employ cryo-electron microscopy to visualize DCTN4's position at the pointed end of the Arp1 filament

• Mutational analysis: Create systematic mutations in DCTN4's cysteine-rich motif, which is known to interact directly with Arp1, and assess binding affinity

• In vitro reconstitution assays: Reconstitute the pointed-end complex with purified components (DCTN4, DCTN5, DCTN6, and ACTR10) to study assembly dynamics

• FRET-based interaction assays: Monitor protein-protein interactions between fluorescently labeled DCTN4 and Arp1 in living cells

• Cross-linking mass spectrometry: Identify specific interaction points between DCTN4 and Arp1

This multi-faceted approach can help elucidate how DCTN4 contributes to pointed-end capping of the Arp1 filament and how this interaction affects dynein-dynactin function in various cellular processes .

What are the critical parameters for validating DCTN4 antibody specificity for Western blot analysis?

When validating a DCTN4 antibody for Western blot applications, researchers should implement the following critical validation steps:

• Positive controls: Include lysates from tissues/cells known to express DCTN4 (such as neuronal cells with active dynein-dependent transport)

• Knockdown controls: Compare DCTN4 detection in samples with and without DCTN4 siRNA/shRNA treatment

• Overexpression controls: Analyze samples with overexpressed tagged DCTN4 to confirm antibody detection at the appropriate molecular weight (approximately 62 kDa)

• Cross-reactivity assessment: Test the antibody against related dynactin subunits to ensure specificity

• Loading controls: Include appropriate housekeeping proteins to normalize expression levels

• Optimization parameters: Determine optimal antibody concentration, incubation time/temperature, and blocking conditions

For DCTN4 polyclonal antibodies, validation across multiple species (human, mouse, rat) should be performed if cross-reactivity is claimed . Additional verification through mass spectrometry identification of immunoprecipitated proteins can provide definitive confirmation of antibody specificity.

What are the optimal storage and handling conditions for maintaining DCTN4 antibody performance?

To ensure optimal performance of DCTN4 antibodies over time:

• Storage temperature: Store concentrated antibody stocks at -20°C as recommended for commercial preparations

• Buffer composition: Typical storage solutions contain phosphate buffered solution (pH 7.4) with stabilizer (0.05%) and glycerol (50%)

• Aliquoting: Divide stock solutions into single-use aliquots to avoid repeated freeze-thaw cycles

• Freeze-thaw minimization: Limit freeze-thaw cycles as they can compromise antibody performance

• Shipping considerations: Upon receipt of shipped antibodies (typically with ice packs), immediately store at the recommended temperature

• Working dilutions: Prepare working dilutions fresh and store at 4°C for short periods only (1-2 weeks)

Following these guidelines helps maintain antibody integrity for up to 12 months as specified by manufacturers . For longer-term storage beyond manufacturer recommendations, validation of antibody performance should be conducted before use in critical experiments.

How should researchers design experiments to study DCTN4 variants associated with disease susceptibility?

When investigating DCTN4 variants associated with disease susceptibility, such as those linked to P. aeruginosa infection in cystic fibrosis:

• Genotyping approach: Implement targeted sequencing or high-resolution melting analysis to identify variants of interest (e.g., rs11954652; Phe349Leu and rs35772018; Tyr270Cys)

• Stratification strategy: Group subjects based on variant burden and conservation status, as different variants may confer varying levels of risk (homozygotes for common variants and heterozygotes for rarer, highly conserved variants may show stronger phenotypes)

• Statistical analysis:

  • Use time-to-event analyses (e.g., Cox proportional hazards models) to assess association with disease outcomes

  • Consider age-dependent hazard ratios as effect sizes may vary with age of onset

• Control for confounding factors: Account for primary disease mutations (e.g., CFTR genotypes) and assess potential interactions

• Functional validation: Complement association studies with cellular assays to determine how variants affect DCTN4 function in relevant processes like autophagy

This comprehensive approach enables robust investigation of the biological mechanisms underlying DCTN4 variant contributions to disease susceptibility .

How can researchers address non-specific binding when using DCTN4 antibodies in immunofluorescence applications?

Non-specific binding in immunofluorescence experiments with DCTN4 antibodies can be minimized through the following strategies:

• Optimization of fixation method: Compare paraformaldehyde, methanol, and acetone fixation to determine which best preserves DCTN4 epitopes while maintaining cellular architecture

• Blocking optimization:

  • Extend blocking time (1-2 hours at room temperature)

  • Test different blocking agents (BSA, normal serum from the secondary antibody host species, commercial blocking solutions)

  • Consider dual blocking with both protein blockers and glycine to quench aldehyde groups

• Antibody dilution adjustment: Test a range of dilutions beyond manufacturer recommendations (typically starting with 1:100-1:300 for IHC applications)

• Background reduction:

  • Include 0.1-0.3% Triton X-100 or 0.05% saponin in blocking and antibody dilution buffers

  • Add 0.05-0.1% Tween-20 in wash buffers

  • Consider including 0.1-0.3M NaCl in antibody dilution buffer to reduce ionic interactions

• Signal validation: Compare staining pattern to expected subcellular localization (punctate cytoplasmic and centrosomal distribution)

When troubleshooting, remember that DCTN4's dual localization pattern (cytoplasmic punctate and centrosomal) should guide your assessment of specific versus non-specific signals.

What strategies can help distinguish between DCTN4 and other dynactin subunits in experimental analyses?

Distinguishing DCTN4 from other dynactin subunits requires careful experimental design:

• Antibody epitope selection: Choose antibodies raised against unique regions of DCTN4 that do not share homology with other dynactin subunits

• Verification techniques:

  • Immunoblotting with recombinant proteins of multiple dynactin subunits

  • Mass spectrometry confirmation of immunoprecipitated proteins

  • Parallel immunostaining with antibodies against multiple dynactin subunits

• Molecular techniques:

  • Subunit-specific knockdown to confirm signal reduction only for the targeted protein

  • Expression of tagged versions of individual subunits to compare localization patterns

• Co-localization analysis: Quantitative co-localization studies with known interaction partners specific to DCTN4 but not other subunits

• Functional assays: Develop readouts that specifically depend on DCTN4's unique functions within the dynactin complex

These approaches help ensure that experimental observations can be confidently attributed to DCTN4 rather than other structurally or functionally related dynactin components.

How can researchers account for potential nuclear localization of DCTN4 in overexpression studies?

The search results indicate that DCTN4 overexpression can lead to both cytosolic and nuclear distribution, suggesting potential nuclear targeting at high expression levels . To account for this phenomenon:

• Expression level control:

  • Use inducible expression systems to titrate DCTN4 levels

  • Monitor expression levels quantitatively using qPCR and Western blotting

  • Compare different promoter strengths to achieve physiological expression

• Subcellular fractionation: Perform nuclear/cytoplasmic fractionation to quantify DCTN4 distribution at different expression levels

• Live-cell imaging: Use photoactivatable or photoconvertible tagged DCTN4 to track protein movement between compartments

• Mutation analysis: Identify and mutate potential nuclear localization signals in DCTN4 to prevent nuclear accumulation

• Functional consequences: Assess whether nuclear localization affects canonical DCTN4 functions or reveals novel nuclear roles

• Physiological relevance: Determine if nuclear localization occurs in endogenous contexts or only under artificial overexpression

Understanding this property of DCTN4 is crucial for accurate interpretation of experimental results, particularly in studies utilizing overexpression approaches .

What emerging techniques might enhance our understanding of DCTN4's role in cellular processes?

Several emerging technologies and approaches hold promise for advancing our understanding of DCTN4 biology:

• Proximity labeling approaches: BioID or APEX2-based approaches can identify proteins in close proximity to DCTN4 in living cells, potentially revealing novel interaction partners

• Super-resolution microscopy: Techniques like STORM, PALM, or STED can resolve DCTN4's precise localization within dynactin complexes at nanoscale resolution

• Cryo-electron tomography: Can provide structural insights into DCTN4's position and conformation within intact dynactin complexes in a near-native state

• Patient-derived cellular models: iPSCs from individuals with DCTN4 variants can be differentiated into relevant cell types to study phenotypic consequences

• Single-molecule tracking: Can reveal dynamics of individual DCTN4-containing complexes in living cells

• Integrative multi-omics approaches: Combining genomics, proteomics, and functional studies to comprehensively understand DCTN4's roles in health and disease

These approaches may help resolve outstanding questions about DCTN4's functions beyond its structural role in dynactin, particularly its potential involvement in autophagy, pathogen defense, and nuclear processes .

How might DCTN4 research inform therapeutic approaches for associated diseases?

Research on DCTN4, particularly its role in P. aeruginosa susceptibility in cystic fibrosis patients, suggests several potential therapeutic avenues:

• Autophagy modulation: Compounds that enhance autophagy might compensate for defective DCTN4-mediated autophagosome transport, potentially improving pathogen clearance

• Gene therapy approaches: Correction of DCTN4 variants in targeted tissues could restore normal dynactin function

• Small molecule screening: Identification of compounds that specifically interact with variant DCTN4 proteins to restore functionality

• Personalized medicine strategies: Genotyping DCTN4 variants to identify high-risk patients who might benefit from earlier or more aggressive antibiotic prophylaxis against P. aeruginosa

• Combination therapies: Targeting both CFTR (the primary cause of cystic fibrosis) and modifier genes like DCTN4 might provide more comprehensive disease management