DTX4 Antibody

What is DTX4 and what cellular functions does it perform?

DTX4 (Deltex 4 E3 ubiquitin ligase) is a member of the Deltex protein family that functions as a regulator of Notch signaling, a pathway involved in cell-cell communications controlling a broad spectrum of cell-fate determinations. In humans, the canonical protein has 619 amino acid residues with a molecular mass of 67.3 kDa and is primarily localized in the cytoplasm. DTX4 is expressed across multiple tissues, including bronchus, thyroid gland, and appendix . Beyond its role in Notch signaling, DTX4 has been identified as a critical component in the negative regulation of type I interferon signaling. It is recruited by NLRP4 (Nod-like receptor protein 4) to facilitate Lys48-linked polyubiquitination and subsequent degradation of TBK1 (TANK-binding kinase 1), thereby maintaining immune homeostasis during antiviral innate immunity .

How many isoforms of DTX4 exist and how do they differ?

Current research indicates that up to two different isoforms of DTX4 have been reported in humans . Most commercially available DTX4 antibodies are designed to detect both isoforms. For instance, the polyclonal antibody described in search result targets a 19 amino acid peptide sequence located between amino acids 230-280 of human DTX4, a region present in both known isoforms. This allows researchers to detect all variants of the protein in their experimental systems. The specific functional differences between these isoforms remain an active area of investigation, particularly regarding tissue-specific expression patterns and potential differential roles in the ubiquitination pathway .

What are the common synonyms and orthologs for DTX4?

DTX4 is known by several synonyms in the scientific literature, which is important to recognize when conducting comprehensive literature searches. These synonyms include E3 ubiquitin-protein ligase DTX4, RING finger protein 155 (RNF155), RING-type E3 ubiquitin transferase DTX4, deltex 4 homolog, and deltex 4 E3 ubiquitin ligase . DTX4 is evolutionarily conserved across multiple species, with orthologs identified in mouse, rat, bovine, frog, chimpanzee, chicken, and fish models. The conservation of this protein across diverse species suggests its fundamental importance in cellular processes and makes comparative studies valuable for understanding its function . Recent studies in fish models have revealed that CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination, demonstrating functional conservation of this regulatory mechanism across vertebrates .

What are the optimal applications for DTX4 antibodies in protein detection?

DTX4 antibodies are validated for multiple experimental applications, with varying optimal concentrations depending on the technique. Western Blot represents one of the most widely used and reliable applications, typically employing DTX4 antibodies at concentrations of 1-2 μg/ml . Immunohistochemistry (IHC) applications generally require slightly higher concentrations, with recommended usage at approximately 5 μg/ml . For immunofluorescence and immunocytochemistry, optimal concentrations are typically higher, around 20 μg/ml to achieve clear signal-to-noise ratios . Additional validated applications include ELISA and flow cytometry, though specific optimization may be required based on experimental conditions and antibody source. When selecting a DTX4 antibody for a specific application, researchers should prioritize those with validation data in their particular application of interest, as performance can vary significantly between experimental contexts .

How should researchers validate the specificity of DTX4 antibodies?

Validating antibody specificity is crucial for obtaining reliable and reproducible research results. For DTX4 antibodies, a multi-faceted validation approach is recommended:

• Western blot analysis using positive control lysates (such as HeLa cells, which express detectable levels of endogenous DTX4)

• Peptide competition assays to confirm binding to the intended epitope

• Knockdown or knockout validation using siRNA or CRISPR approaches targeting DTX4

• Cross-reactivity testing against other DTX family members (DTX1, DTX2, DTX3) to ensure specificity

Most commercial DTX4 antibodies are predicted not to cross-react with other DTX protein family members, but experimental validation is still essential . When performing knockdown experiments, researchers have successfully used NLRP4-specific siRNA and NLRP4-specific lentivirus shRNA constructs to assess the functional relationship between NLRP4 and DTX4 in TBK1 degradation, providing a useful methodological framework for similar studies focused on DTX4 directly .

What is the recommended sample preparation protocol for DTX4 detection in Western blot?

For optimal detection of DTX4 protein in Western blot applications, researchers should follow this methodological approach:

• Cell lysis should be performed using a buffer containing appropriate protease inhibitors and preferably phosphatase inhibitors (especially when studying ubiquitination dynamics)

• Include 1% NP-40 or Triton X-100 to effectively solubilize membrane-associated proteins

• Use fresh samples whenever possible, as freeze-thaw cycles can affect protein integrity

• Load 20-50 μg of total protein per lane for cell lysates

• For SDS-PAGE, use 10% gels to achieve optimal resolution around the 68 kDa range (theoretical molecular weight of DTX4)

• Transfer to PVDF membranes (rather than nitrocellulose) for improved protein retention

• Block with 5% BSA rather than milk, as milk can interfere with phospho-specific detection

• Use DTX4 antibody at 1-2 μg/ml concentration with overnight incubation at 4°C

It's important to note that the observed molecular weight may differ from the predicted 68 kDa due to post-translational modifications, cleavage, or isoform expression . When studying the interaction between DTX4 and other proteins like TBK1, co-immunoprecipitation protocols may be necessary, as demonstrated in studies of the NLRP4-DTX4-TBK1 axis .

How does DTX4 regulate type I interferon signaling?

DTX4 functions as a negative regulator of type I interferon signaling through its E3 ubiquitin ligase activity targeting TBK1. The regulatory pathway involves several steps: First, the pattern-recognition receptor NLRP4 recognizes specific molecular patterns and subsequently recruits DTX4 to TBK1. Then, DTX4 catalyzes the Lys48 (K48)-linked polyubiquitination of TBK1, specifically at residue Lys670, which targets TBK1 for proteasomal degradation . This degradation prevents excessive TBK1 activation, thereby limiting downstream phosphorylation of the transcription factor IRF3 and type I interferon production.

The importance of this regulatory mechanism has been demonstrated through knockdown experiments. When either DTX4 or NLRP4 is depleted using siRNA, the K48-linked ubiquitination and degradation of TBK1 are significantly reduced, resulting in enhanced phosphorylation of TBK1 and IRF3 . This leads to increased interferon-responsive gene expression and enhanced antiviral immunity, as demonstrated by resistance to viral infection in cells with NLRP4 knockdown . This negative regulatory function represents a critical mechanism for maintaining immune homeostasis during antiviral responses.

What is the relationship between DTX4 and the Notch signaling pathway?

DTX4, as a member of the Deltex protein family, is involved in the regulation of Notch signaling, although its precise function appears to be complex and context-dependent. The Notch pathway is a highly conserved signaling system that regulates cell fate decisions through direct cell-cell communication . DTX proteins typically contain N-terminal Notch-binding domains and C-terminal RING finger domains with E3 ubiquitin ligase activity, allowing them to influence Notch receptor processing and stability through ubiquitination.

While DTX4's exact role in Notch signaling is still being elucidated, by similarity to other Deltex family members, it likely functions as a modulator of Notch receptor activity through ubiquitination-dependent mechanisms. This function may vary across different tissue contexts, developmental stages, and in response to different cellular stressors. The dual role of DTX4 in both Notch signaling and innate immune responses suggests interesting cross-talk between these pathways that may be particularly relevant in tissues with high DTX4 expression, such as bronchus, thyroid gland, and appendix . Further research using tissue-specific knockdown approaches could help clarify the tissue-specific functions of DTX4 in Notch signaling.

How do recent findings about DTX4-mediated TBK1 degradation in fish relate to mammalian systems?

Recent research has revealed that in fish models, CDK2 (Cyclin-dependent kinase 2) recruits Dtx4 to facilitate the degradation of TBK1 through ubiquitination . This finding extends our understanding of DTX4 function across species and suggests an additional regulatory mechanism controlling DTX4-mediated ubiquitination of TBK1. The study demonstrated that overexpression of DTX4 amplified CDK2-mediated inhibition of interferon promoter activity induced by TBK1 . Additionally, DTX4 overexpression increased TBK1 degradation beyond what was observed with CDK2 alone, while DTX4 knockdown significantly diminished CDK2 suppression of TBK1-induced interferon promoter activity .

These findings from fish models suggest a potentially conserved mechanism where cell cycle regulators (CDK2) interface with innate immune responses through DTX4-mediated regulation of TBK1. While direct evidence in mammalian systems is still emerging, the conservation of these proteins across species suggests this pathway may be relevant in human contexts as well. This represents an important area for further research, particularly examining whether human CDK2 interacts with DTX4 to regulate TBK1 stability during viral infections or other immune challenges. Comparative studies between fish and mammalian systems could provide valuable insights into the evolutionary conservation of this regulatory mechanism .

What criteria should guide selection of DTX4 antibodies for specific experimental purposes?

When selecting DTX4 antibodies for research applications, several critical factors should be considered based on the intended experimental use:

For advanced research into DTX4's role in ubiquitination pathways, select antibodies that have been validated in ubiquitination assays or that don't interfere with the E3 ligase activity of DTX4. Additionally, consider whether the antibody recognizes particular domains of interest (such as the RING domain) when studying specific protein functions .

What are common troubleshooting approaches for failed DTX4 detection in Western blot?

When troubleshooting failed detection of DTX4 in Western blot experiments, researchers should systematically address these potential issues:

• Protein extraction efficiency: DTX4 is primarily cytoplasmic, but may associate with membrane components or protein complexes. Use extraction buffers containing 1% NP-40 or Triton X-100 to ensure efficient solubilization.

• Protein degradation: DTX4 may be susceptible to degradation during sample preparation. Include both protease inhibitors and deubiquitinase inhibitors (such as N-ethylmaleimide) in lysis buffers.

• Detection sensitivity: If endogenous levels are low, consider:

  • Increasing protein loading (50-80 μg per lane)

  • Using enhanced chemiluminescence detection systems

  • Performing enrichment through immunoprecipitation before Western blot

• Antibody specificity issues: Some antibodies may recognize only specific isoforms or may be affected by post-translational modifications. Try antibodies targeting different epitopes of DTX4.

• Transfer efficiency: Proteins around 68 kDa may require optimized transfer conditions. Consider longer transfer times or lower methanol concentrations in transfer buffer .

• Sample type: Ensure your cell/tissue model expresses detectable levels of DTX4. HeLa cells have been documented to express DTX4 at detectable levels for Western blot applications .

If detection continues to be problematic, consider using overexpression systems as positive controls to verify antibody functionality before proceeding with endogenous detection attempts.

How can researchers optimize immunofluorescence protocols for DTX4 detection?

Optimizing immunofluorescence (IF) protocols for DTX4 detection requires attention to several methodological details:

• Fixation: Compare paraformaldehyde (4%) with methanol fixation to determine which better preserves DTX4 epitopes while maintaining cellular architecture.

• Permeabilization: For cytoplasmic proteins like DTX4, use 0.1-0.2% Triton X-100 for 10 minutes at room temperature for optimal antibody access to intracellular epitopes.

• Antibody concentration: DTX4 detection typically requires higher antibody concentrations in IF compared to Western blot. Start with 20 μg/ml and adjust based on signal intensity and background .

• Incubation conditions: Extend primary antibody incubation to overnight at 4°C to enhance specific binding while reducing background.

• Signal amplification: Consider using tyramide signal amplification systems for low-abundance targets.

• Controls: Include appropriate controls such as:

  • Peptide competition to confirm specificity

  • DTX4 knockdown samples as negative controls

  • Cells with known DTX4 expression patterns as positive controls

  • Secondary antibody-only controls to assess background

• Counterstaining: Use appropriate subcellular markers (such as cytoskeletal or organelle markers) to confirm the expected cytoplasmic localization pattern of DTX4.

Human spleen tissue has been documented as suitable for immunofluorescence detection of DTX4, with successful staining achieved using DTX4 antibody at 20 μg/ml concentration .

How can researchers effectively study DTX4-mediated ubiquitination of target proteins?

Studying DTX4-mediated ubiquitination requires specialized experimental approaches:

• In vitro ubiquitination assays:

  • Purify recombinant DTX4 protein with intact RING domain

  • Include purified E1 (ubiquitin-activating) and E2 (ubiquitin-conjugating) enzymes

  • Add purified substrate (e.g., TBK1) and ubiquitin (consider using tagged ubiquitin for easier detection)

  • Analyze reaction products by Western blot using anti-ubiquitin antibodies

• Cell-based ubiquitination studies:

  • Co-express DTX4, target substrate (e.g., TBK1), and HA-tagged ubiquitin

  • Treat cells with proteasome inhibitors (MG132) to prevent degradation of ubiquitinated proteins

  • Immunoprecipitate the substrate protein

  • Perform Western blot with anti-HA antibodies to detect ubiquitination

• Analysis of ubiquitin chain topology:

  • Use ubiquitin mutants (K48R, K63R) to determine the type of ubiquitin chains

  • Alternatively, use linkage-specific anti-ubiquitin antibodies

  • For DTX4-mediated TBK1 ubiquitination, focus on K48-linked chains which target proteins for degradation

• Mapping ubiquitination sites:

  • Generate substrate mutants (e.g., TBK1 K670R) to confirm specific ubiquitination sites

  • Perform mass spectrometry analysis of purified ubiquitinated proteins to identify modified residues

The NLRP4-DTX4-TBK1 axis study provides an excellent methodological framework, demonstrating that NLRP4 recruits DTX4 for K48-linked polyubiquitination of TBK1 at Lys670, which leads to its degradation .

What experimental approaches can elucidate the role of DTX4 in viral infection responses?

To investigate DTX4's role in viral infection responses, researchers can employ several sophisticated experimental approaches:

• Gene silencing and overexpression strategies:

  • Use siRNA or shRNA targeting DTX4 in relevant cell types

  • Create stable DTX4 knockdown cell lines using lentiviral shRNA constructs

  • Generate DTX4-overexpressing cells to observe effects on antiviral responses

  • Measure type I interferon production, viral replication, and cell survival

• Viral infection models:

  • Challenge cells with RNA viruses (e.g., vesicular stomatitis virus [VSV]) that strongly activate TBK1-dependent pathways

  • Quantify viral replication using plaque assays, qPCR, or fluorescent reporter viruses

  • Analyze the expression of interferon-stimulated genes using qRT-PCR or RNA-seq

• Signaling pathway analysis:

  • Monitor TBK1 and IRF3 phosphorylation status by Western blot

  • Use luciferase reporter assays driven by interferon-responsive elements (ISRE)

  • Analyze the dynamics of NLRP4-DTX4-TBK1 complex formation during infection

• In vivo models:

  • Generate DTX4 conditional knockout mice

  • Challenge with viral infections and monitor survival, viral loads, and immune responses

  • Analyze tissue-specific effects in organs with high DTX4 expression

Studies have demonstrated that knockdown of NLRP4 (which recruits DTX4) renders cells more resistant to viral infection with significantly fewer virus-infected cells compared to control conditions . Similar approaches targeting DTX4 directly would help elucidate its specific contribution to antiviral immunity regulation.

How does the interaction between CDK2, DTX4, and TBK1 influence cell cycle regulation and immune responses?

The newly discovered relationship between CDK2, DTX4, and TBK1 represents an intriguing intersection between cell cycle regulation and immune responses. To investigate this complex interplay, researchers should consider these experimental approaches:

• Protein-protein interaction studies:

  • Perform co-immunoprecipitation experiments to verify CDK2-DTX4 and DTX4-TBK1 interactions

  • Use proximity ligation assays to visualize these interactions in situ

  • Employ FRET or BiFC techniques to monitor dynamic interactions in living cells

• Cell cycle synchronization experiments:

  • Synchronize cells at different cell cycle phases and assess DTX4-mediated TBK1 degradation

  • Determine if viral infection alters CDK2 activity and subsequent DTX4 recruitment

  • Evaluate whether cell cycle inhibitors affect DTX4-mediated immune regulation

• Phosphorylation analysis:

  • Investigate whether CDK2 phosphorylates DTX4 to regulate its E3 ligase activity

  • Identify potential phosphorylation sites by mass spectrometry

  • Generate phospho-mimetic and phospho-deficient DTX4 mutants to assess functional consequences

• Transcriptomic and functional analyses:

  • Perform RNA-seq in cells with manipulated CDK2, DTX4, or TBK1 levels

  • Analyze interferon-stimulated gene expression across the cell cycle

  • Assess viral susceptibility in relation to cell cycle phase and CDK2 activity

Recent findings in fish models have shown that CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination, and overexpression of DTX4 amplifies CDK2-mediated inhibition of interferon promoter activity . Extending these studies to mammalian systems would provide valuable insights into potential evolutionary conservation of this regulatory mechanism and its implications for understanding the coordination between cell proliferation and immune defense.