DTX3L Antibody

What is DTX3L and why is it studied in research?

DTX3L (deltex E3 ubiquitin ligase 3L) is a multifunctional protein that acts as an E3 ubiquitin ligase. It is also known by several alternative names including BBAP, RNF143, and E3 ubiquitin-protein ligase DTX3L. The protein has a molecular weight of approximately 83.6 kilodaltons . DTX3L has gained significant research interest due to its roles in:

• DNA damage repair pathways

• Interferon-mediated antiviral responses

• Regulation of tumor cell proliferation and drug resistance

• Histone modification through ubiquitination

• Complex formation with PARP9 to regulate multiple cellular processes

Research on DTX3L has expanded significantly with its implications in cancer biology, viral infection responses, and DNA repair mechanisms, making DTX3L antibodies essential tools for investigating these critical biological processes .

What applications are DTX3L antibodies commonly used for?

DTX3L antibodies are utilized across multiple experimental techniques to study its expression, localization, and functional interactions. The primary applications include:

Multiple validated antibodies are available from suppliers, with applications specifically documented for human, mouse, and rat samples .

How does DTX3L function in the antiviral immune response?

DTX3L plays a critical role in antiviral immunity through multiple mechanisms:

DTX3L establishes an IFN-β–ETS1–DTX3L–TBK1 positive-feedback loop that enhances interferon signaling during viral infection. This pathway functions through:

• Viral infection triggering initial type I interferon production

• Type I interferon promoting ETS1 translocation to the nucleus

• ETS1 enhancing DTX3L promoter activity and expression

• DTX3L ubiquitinating TBK1 at K30 and K401 sites via K63-linked ubiquitination

• DTX3L mediating TBK1 phosphorylation through interaction with SRC tyrosine kinase

• Activated TBK1 further amplifying interferon production

This feedback mechanism significantly restricts viral replication, as demonstrated through loss-of-function experiments. When DTX3L is knocked down or knocked out in cells, RSV (respiratory syncytial virus) titers increase substantially, while the expression of interferon-stimulated genes (ISGs) such as OAS1, ISG15, and IFIT1 decreases .

Additionally, DTX3L works in concert with PARP9 to target viral proteins directly for degradation through K48-linked ubiquitination, as shown with encephalomyocarditis virus (EMCV) and human rhinovirus (HRV) C3 proteases .

How should researchers optimize Western blotting protocols for DTX3L detection?

Optimizing Western blotting for DTX3L requires specific considerations due to its molecular weight and expression patterns:

Protocol Optimization Recommendations:

• Sample preparation:

  • Use RIPA buffer supplemented with protease inhibitors and deubiquitinase inhibitors (N-ethylmaleimide at 10mM)

  • Include phosphatase inhibitors when studying DTX3L phosphorylation

  • Sonicate samples briefly (3 x 10s pulses) to ensure complete lysis

• Gel selection and separation:

  • Use 8% SDS-PAGE gels for optimal separation around 83.6 kDa

  • Run gels at lower voltage (80-100V) for better resolution

• Transfer conditions:

  • Transfer at 100V for 2 hours in cold room or 30V overnight

  • Use PVDF membranes rather than nitrocellulose for better protein retention

• Blocking and antibody incubation:

  • Block with 5% non-fat milk in TBST for standard applications

  • For phospho-specific detection, use 5% BSA in TBST

  • Incubate primary antibodies (1:1000 dilution) overnight at 4°C

  • Wash extensively (4 x 10 minutes) with TBST before secondary antibody

• Controls and validation:

  • Include positive controls (cells with known DTX3L expression)

  • Use DTX3L knockout or knockdown samples as negative controls

  • Validate specificity with competing peptides when possible

When analyzing results, note that DTX3L expression may be induced by interferon treatment or DNA damage, which can serve as positive controls for antibody validation .

What is the relationship between DTX3L and DNA damage repair, and how can researchers study this interaction?

DTX3L plays a critical role in DNA double-strand break (DSB) repair through multiple mechanisms that can be investigated using specialized techniques:

Key DTX3L Functions in DNA Repair:

• DTX3L forms a regulatory axis with the deubiquitinase USP28 at DNA DSBs:

  • DTX3L ubiquitinates USP28, targeting it for proteasomal degradation

  • USP28 counteracts by deubiquitinating both itself and DTX3L

  • This cross-regulation fine-tunes DSB repair across multiple pathways

• DTX3L, together with PARP9, mediates histone modifications critical for repair:

  • Monoubiquitinates histone H4 at Lys-91 (H4K91ub1)

  • This modification serves as a licensing signal for subsequent histone H4 post-translational modifications

  • Promotes recruitment of repair factors including 53BP1, RAP80, and BRCA1 to DNA damage sites

• DTX3L regulates TIRR nuclear export and degradation:

  • Ubiquitinates TIRR at lysine 187

  • Facilitates XPO1-mediated TIRR nuclear export

  • This regulates the balance between NHEJ and HR repair pathways

Experimental Approaches to Study DTX3L in DNA Repair:

When designing experiments, consider that DTX3L effects on repair may be cell-type specific and depend on its interaction with PARP9, USP28, and other factors. The complex cross-regulation between DTX3L and USP28 suggests that knockdown of either protein alone may give misleading results - double knockdown experiments may be necessary to fully understand their functional relationship .

How does DTX3L regulate TIRR and 53BP1 in the context of DNA repair pathway choice?

DTX3L plays a sophisticated role in regulating the choice between non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways through its effects on the TIRR-53BP1 axis:

Mechanism of DTX3L-mediated regulation:

• DTX3L ubiquitinates Tudor Interacting Repair Regulator (TIRR) at lysine 187

• This ubiquitination facilitates two critical processes:

  • XPO1-mediated nuclear export of TIRR to the cytoplasm

  • Degradation of TIRR following DNA damage

• Reduced nuclear TIRR releases 53BP1 from TIRR-mediated inhibition

• Activated 53BP1 promotes NHEJ repair and inhibits HR repair

• This pathway makes cells more sensitive to PARP inhibitors by inducing HR deficiency

Research findings with clinical implications:

DTX3L is frequently overexpressed in prostate cancers. This overexpression leads to:

• Decreased TIRR levels

• Impaired negative regulation of 53BP1

• Induction of HR deficiency

• Increased chromosomal instability

• Enhanced sensitivity to PARP inhibitors

These findings suggest that DTX3L overexpression could serve as a biomarker for predicting PARP inhibitor sensitivity in cancers, particularly prostate cancer .

Experimental approaches to study this pathway:

• Immunofluorescence to monitor 53BP1 foci formation in response to DNA damage

• Subcellular fractionation to track TIRR nuclear export

• Ubiquitination assays to detect DTX3L-mediated TIRR modification

• PARP inhibitor sensitivity assays in cells with modulated DTX3L expression

• Chromatin immunoprecipitation to assess 53BP1 binding to damaged chromatin

When designing these experiments, researchers should consider the timing of measurements, as the dynamics of TIRR nuclear export and degradation may vary depending on the type and severity of DNA damage .

What are common issues in DTX3L detection and how can they be resolved?

Researchers working with DTX3L antibodies may encounter several technical challenges. Here are common issues and recommended solutions:

Problem 1: Weak or no signal in Western blotting

• Potential causes:

  • Low DTX3L expression in unstimulated cells

  • Inadequate extraction of nuclear proteins

  • Antibody epitope masking due to protein-protein interactions

• Solutions:

  • Stimulate cells with IFNγ or DNA-damaging agents to upregulate DTX3L

  • Use nuclear extraction protocols with high salt buffers (≥400mM NaCl)

  • Try different antibodies targeting distinct DTX3L epitopes

  • Increase protein loading (50-100μg total protein)

  • Extend primary antibody incubation to overnight at 4°C

Problem 2: Multiple bands or non-specific binding

• Potential causes:

  • Cross-reactivity with other DTX family members

  • Detection of post-translationally modified forms

  • Non-specific binding

• Solutions:

  • Use DTX3L knockout/knockdown controls to identify specific bands

  • Run gradient gels to better separate closely migrating bands

  • Increase washing stringency with higher detergent concentration

  • Pre-absorb antibody with recombinant DTX1 or DTX2 proteins

  • Validate with an antibody targeting a different region of DTX3L

Problem 3: Inconsistent immunoprecipitation results

• Potential causes:

  • Dynamic interactions dependent on cellular conditions

  • Epitope masking in protein complexes

  • Incompatible lysis conditions

• Solutions:

  • Cross-link protein interactions before lysis

  • Test multiple lysis buffers (RIPA vs. NP-40 vs. digitonin)

  • Try native vs. denaturing conditions

  • Use tag-based systems for pulling down DTX3L complexes

  • Consider proximity ligation assays as an alternative approach

For immunofluorescence applications, permeabilization conditions are critical - use 0.5% Triton X-100 for 10 minutes to ensure adequate nuclear penetration while preserving DTX3L epitopes .

How can researchers validate DTX3L antibody specificity for their experimental system?

Thorough validation of DTX3L antibodies is essential for reliable research outcomes. This multi-faceted approach ensures specificity and appropriate application:

Comprehensive Validation Strategy:

• Genetic validation approaches:

  • Compare signals between wild-type and DTX3L knockout cells

  • Use DTX3L siRNA/shRNA knockdown cells (reference shows shRNA1 effectively reduces DTX3L levels)

  • Perform rescue experiments with DTX3L overexpression constructs

• Biochemical validation:

  • Test reactivity against recombinant DTX3L protein

  • Peptide competition assays using the immunizing peptide

  • Compare multiple antibodies targeting different DTX3L epitopes

• Application-specific validation:

  • For Western blotting: Verify band at expected molecular weight (~83.6 kDa)

  • For IHC/ICC: Include peptide blocking controls

  • For IP: Confirm enrichment by Western blotting

  • For all applications: Include IFNγ-stimulated vs. unstimulated controls

• Cross-reactivity assessment:

  • Test against recombinant DTX1, DTX2, and DTX4 proteins

  • Compare patterns in cells expressing different DTX family members

  • Evaluate species cross-reactivity if working with non-human models

DTX3L antibody validation table:

When publishing research using DTX3L antibodies, document validation methods thoroughly and include representative images of controls to establish antibody specificity .

How can researchers effectively study the DTX3L-PARP9 complex in antiviral responses?

The DTX3L-PARP9 complex plays a significant role in antiviral immunity through multiple mechanisms. Here are specialized approaches to study this complex:

Experimental Strategies:

• Complex formation and stability:

  • Co-immunoprecipitation with antibodies against either DTX3L or PARP9

  • Size exclusion chromatography to isolate the native complex

  • FRET or BiFC assays to monitor interaction in living cells

  • Native PAGE to preserve complex integrity

• Functional analysis in antiviral responses:

  • Compare viral replication in cells with DTX3L knockout, PARP9 knockout, or double knockout

  • Use domain mutants to identify regions required for complex function

  • Monitor interferon-stimulated gene expression in response to complex manipulation

  • Assess K63-linked vs. K48-linked ubiquitination patterns

• ADP-ribosylation analysis:

  • Use GST-Af1521 pulldowns to detect ADP-ribosylated proteins (use G42E mutant as control)

  • Apply PARP14 inhibitors to distinguish between different PARP-mediated modifications

  • Detect mono-ADP-ribosylation using AbD43647 reagent

  • Compare ADP-ribosylation patterns with and without IFNγ stimulation

Key insights from recent research:

A study demonstrated that PARP14 is regulated by the PARP9/DTX3L complex, with these proteins forming an intricate regulatory network in interferon responses. Researchers showed that:

• DTX3L and PARP14 are ADP-ribosylated under basal conditions

• IFNγ treatment substantially increases this ADP-ribosylation

• PARP14 inhibitors revert the IFNγ-induced ADP-ribosylation

• In DTX3L knockout cells, IFNγ-induced ADP-ribosylation of PARP14 is reduced

This suggests PARP14 promotes both auto-ADP-ribosylation and DTX3L trans-ADP-ribosylation in response to IFNγ, highlighting the complex cross-regulation within this system .

When designing experiments to study this complex, consider the timing of stimulation, as IFNγ-induced effects typically peak after 12-24 hours of treatment .

How does DTX3L influence cell adhesion and drug resistance in cancer cells?

DTX3L has emerged as a significant factor in cancer biology, particularly regarding cell adhesion-mediated drug resistance (CAM-DR) in multiple myeloma (MM) and other cancers:

DTX3L's role in cancer cell biology:

• Cell proliferation effects:

  • DTX3L promotes MM cell proliferation

  • Silencing DTX3L results in G1 phase cell cycle arrest

  • This corresponds with decreased expression of cyclin E and CDK2

  • This effect may be cell-type specific and context-dependent

• Cell adhesion regulation:

  • DTX3L expression increases when MM cells adhere to fibronectin (FN) or HS-5 stromal cells

  • Knockdown of DTX3L significantly reduces cell adhesion rates

  • This suggests DTX3L mediates interactions between MM cells and the bone marrow microenvironment

• Drug resistance mechanisms:

  • DTX3L contributes to cell adhesion-mediated drug resistance (CAM-DR)

  • MM cells become more sensitive to chemotherapy drugs after DTX3L silencing

  • DTX3L is regulated by the focal adhesion kinase (FAK) signaling pathway

  • This creates a mechanism where adhesion signals promote drug resistance via DTX3L

Research approaches to study DTX3L in cancer:

These findings suggest that targeting DTX3L could potentially overcome drug resistance in MM and other cancers by disrupting the adhesion-mediated protective effects of the tumor microenvironment .

What is the functional relationship between DTX3L and USP28 in DNA repair, and how can researchers investigate this interplay?

The relationship between DTX3L (an E3 ubiquitin ligase) and USP28 (a deubiquitinase) represents a sophisticated regulatory mechanism in DNA repair that can be investigated through specialized approaches:

Molecular mechanisms of the DTX3L-USP28 regulatory axis:

• Antagonistic activities:

  • DTX3L ubiquitinates USP28, leading to its proteasomal degradation

  • USP28 deubiquitinates both itself and DTX3L, counteracting DTX3L's effect

  • This creates a dynamic feedback loop that calibrates DNA repair responses

• Structural basis of interaction:

  • The N-terminal D1-D3 domains of DTX3L primarily mediate interaction with USP28

  • DTX3L and USP28 physically interact and colocalize in cellular sub-compartments

  • This interaction is enhanced following DNA damage

• Impact on repair pathways:

  • The DTX3L-USP28 circuit influences multiple DSB repair pathways:

    • Non-homologous end joining (NHEJ)

    • Homologous recombination (HR)

    • Single-strand annealing (SSA)

    • Microhomology-mediated end joining (MMEJ)

  • Notably, USP28 depletion's detrimental effects on these repair pathways can be rescued by concurrent DTX3L knockdown

Research methods to study this interaction:

• Protein-protein interaction analysis:

  • Co-immunoprecipitation under different DNA damage conditions

  • Domain mapping using truncation mutants

  • Proximity ligation assays in intact cells

  • FRET-based interaction studies

• Ubiquitination/deubiquitination assays:

  • In vitro ubiquitination assays with purified components

  • Cell-based ubiquitination assays following DNA damage

  • Ubiquitin chain linkage analysis (K48 vs. K63)

  • Proteasome inhibition studies to stabilize ubiquitinated forms

• Functional DNA repair assays:

  • DSB repair reporter assays for different pathways

  • Ionizing radiation survival curves

  • Laser microirradiation combined with live-cell imaging

  • Genetic interaction studies with double knockdowns/knockouts

When designing experiments, consider that the DTX3L-USP28 circuit also influences levels of key stress response proteins like HIF-1α, p53, and c-MYC, suggesting broader impacts beyond direct DNA repair that may need to be accounted for in experimental design .

How can researchers utilize DTX3L antibodies to study its role in the regulation of TBK1 and antiviral signaling?

DTX3L plays a critical role in regulating TANK-binding kinase 1 (TBK1) and antiviral signaling pathways. Researchers can employ several sophisticated approaches using DTX3L antibodies to investigate these mechanisms:

Key aspects of DTX3L-TBK1 regulation:

• Ubiquitination-mediated activation:

  • DTX3L ubiquitinates TBK1 at specific lysine residues (K30 and K401)

  • This utilizes K63-linked ubiquitination, which is non-degradative

  • This modification enhances TBK1 activity rather than targeting it for degradation

• Phosphorylation regulation:

  • DTX3L mediates TBK1 phosphorylation by facilitating interaction with SRC tyrosine kinase

  • This phosphorylation is critical for TBK1 activation

  • The process enhances IRF3 dimerization and subsequent interferon production

• Feedback amplification:

  • Type I interferon receptor signaling increases DTX3L expression via ETS1

  • DTX3L then enhances TBK1 activity

  • This creates a positive feedback loop that amplifies antiviral responses

Experimental approaches using DTX3L antibodies:

• Protein modification analysis:

  • Immunoprecipitate TBK1 and blot for ubiquitin to detect DTX3L-mediated modifications

  • Use phospho-specific antibodies to monitor TBK1 activation status

  • Employ native PAGE to assess IRF3 dimerization downstream of TBK1

  • Compare conditions with and without viral infection or poly(I:C) stimulation

• Protein interaction studies:

  • Co-immunoprecipitation of DTX3L and TBK1 under different stimulation conditions

  • Immunofluorescence to visualize DTX3L-TBK1 colocalization during infection

  • Proximity ligation assays to confirm direct interaction in intact cells

  • Use domain mutants to map interaction regions

• Functional signaling analysis:

  • Compare interferon production in WT vs. DTX3L-deficient cells

  • Monitor ISG (OAS1, ISG15, IFIT1) expression as readouts of signaling

  • Use IFNAR1 blocking antibodies to distinguish direct vs. feedback effects

  • Measure viral replication as a functional outcome

Research insights from experimental data:

Research has demonstrated that the interaction between endogenous DTX3L and TBK1 is enhanced upon viral infection (RSV) and poly(I:C) stimulation. Downregulation of DTX3L decreased TBK1 phosphorylation and IRF3 dimerization, while not affecting other signaling components like RIG-I, MAVS, p65, p38, or their phosphorylation, highlighting the specificity of DTX3L's role in the TBK1-IRF3 axis .

When designing these experiments, researchers should consider the kinetics of the response, as DTX3L-TBK1 interactions are typically enhanced within 1-4 hours of stimulation and may vary depending on the stimulus used .

What are the emerging applications of DTX3L antibodies in cancer and antiviral research?

DTX3L antibodies are becoming increasingly valuable tools in several cutting-edge research areas, with significant implications for both cancer biology and antiviral immunity:

Emerging cancer research applications:

• PARP inhibitor sensitivity biomarkers:

  • DTX3L overexpression correlates with reduced TIRR levels and HR deficiency

  • This creates synthetic lethality with PARP inhibitors

  • DTX3L antibodies can help identify tumors likely to respond to PARP inhibitor therapy

  • This is particularly relevant in prostate cancer, where DTX3L is frequently overexpressed

• Cell adhesion-mediated drug resistance:

  • DTX3L mediates interactions between cancer cells and their microenvironment

  • Antibodies can be used to study how these interactions promote chemotherapy resistance

  • This may reveal new therapeutic targets to overcome CAM-DR in multiple myeloma and other cancers

• DNA repair pathway profiling:

  • The DTX3L-USP28 regulatory circuit affects multiple DNA repair pathways

  • Antibodies can help characterize repair deficiencies in tumor samples

  • This may guide personalized therapy based on specific repair defects

Antiviral research frontiers:

• Viral pathogenesis mechanisms:

  • DTX3L restricts RSV replication through interferon amplification

  • Antibodies can track DTX3L-TBK1 interactions during infection

  • This may reveal virus-specific differences in interferon evasion strategies

• Direct antiviral activities:

  • DTX3L/PARP9 complex targets viral proteases for degradation

  • Antibodies can help characterize this novel mechanism across different viral families

  • This may identify vulnerable viral proteins for therapeutic targeting

• Cross-regulation with ADP-ribosylation:

  • DTX3L interacts with PARP9 and PARP14 in complex regulatory networks

  • Antibodies can map these interactions and their modulation during infection

  • This may reveal new targets for antiviral intervention

Future research directions:

The continued development and characterization of highly specific DTX3L antibodies will enable deeper investigation of:

• Tissue-specific and context-dependent functions of DTX3L

• Dynamic regulation of DTX3L during disease progression

• Potential therapeutic targeting of DTX3L in cancer and viral infections

• Cross-talk between DNA repair and antiviral signaling pathways

These applications highlight the growing importance of DTX3L antibodies as tools for both basic research and translational medicine .

What key considerations should researchers keep in mind when designing experiments with DTX3L antibodies?

When designing experiments with DTX3L antibodies, researchers should consider several critical factors to ensure robust and reproducible results:

Experimental design considerations:

• Antibody selection and validation:

  • Choose antibodies with validated specificity for the intended application

  • Verify DTX3L detection in your specific experimental system

  • Include appropriate positive controls (IFNγ-stimulated cells) and negative controls (DTX3L knockdown/knockout)

  • Consider using multiple antibodies targeting different epitopes for confirmation

• Context-dependent expression:

  • DTX3L expression varies significantly based on cellular conditions:

    • Upregulated by type I and II interferons

    • Increased following DNA damage

    • Enhanced during viral infection

    • Elevated in certain cancer types

  • Design experiments with appropriate baseline and stimulated conditions

• Functional interactions:

  • DTX3L functions in complex with multiple partners:

    • PARP9 in antiviral responses and DNA repair

    • USP28 in DNA repair regulation

    • TBK1 in interferon signaling

    • TIRR in repair pathway choice

  • Consider co-immunoprecipitation or proximity ligation assays to verify relevant interactions

• Post-translational modifications:

  • DTX3L itself undergoes modifications:

    • ADP-ribosylation (enhanced by IFNγ)

    • Ubiquitination (regulated by USP28)

    • Potential phosphorylation

  • These modifications may affect antibody recognition and function

Technical optimization strategies:

Data interpretation considerations:

When analyzing results from DTX3L antibody experiments, researchers should:

• Consider the dynamic and feedback-regulated nature of DTX3L expression

• Recognize that DTX3L functions may be cell-type and context-specific

• Account for the complex interplay between ubiquitination and ADP-ribosylation pathways

• Interpret results in light of DTX3L's multiple roles in DNA repair, antiviral immunity, and cell adhesion