DTX3 Antibody

What is DTX3 and what is its biological significance?

DTX3, also known as Deltex3 or RING finger protein 154, is a 347 amino acid protein containing a RING-type zinc finger domain characteristic of the Deltex family. The protein functions primarily as a regulator of Notch signaling, a critical pathway involved in cell-cell communications that governs a broad spectrum of cell-fate determinations . At the molecular level, DTX3 has been identified as an E3 ubiquitin ligase that promotes the ubiquitination and degradation of NOTCH2 . This regulatory function positions DTX3 as a potentially significant player in developmental processes and disease pathways, particularly in cancer where Notch signaling is frequently dysregulated.

The importance of DTX3 has been highlighted in research showing that it acts as an anti-oncogene in esophageal carcinoma, demonstrating a significant negative correlation with NOTCH2 expression in human esophageal cancer samples . The DTX3-NOTCH2 axis represents a promising area for therapeutic intervention in certain cancer types.

What cellular localization pattern does DTX3 exhibit?

DTX3 demonstrates a complex subcellular distribution pattern that researchers should consider when designing experiments. Immunofluorescence and cellular fractionation studies indicate that DTX3 is present in both the nucleus and cytoplasm of cells, with a predominant localization in the nucleus . In contrast, its substrate NOTCH2 is primarily localized in the cytoplasm, with interaction between these proteins occurring in the cytoplasmic compartment .

When conducting immunofluorescence studies with DTX3 antibodies, researchers should optimize fixation and permeabilization protocols to ensure adequate access to both nuclear and cytoplasmic compartments. Co-localization studies with NOTCH2 are particularly informative and should focus on the cytoplasmic region where functional interactions occur.

What criteria should guide the selection of a DTX3 antibody for specific applications?

When selecting a DTX3 antibody, researchers should consider several critical parameters based on their experimental requirements:

• Target epitope specificity: Different antibodies target distinct regions of DTX3, such as AA 201-301 or AA 90-347 . Select antibodies that target conserved regions for cross-species studies or functional domains for mechanistic investigations.

• Host species and clonality: Available options include rabbit polyclonal and mouse monoclonal antibodies . Monoclonal antibodies offer higher specificity but may recognize fewer epitopes, while polyclonal antibodies provide stronger signals but potentially more background.

• Validated applications: Ensure the antibody has been validated for your specific application (WB, IHC, ELISA, etc.) . Cross-application performance cannot be assumed.

• Species reactivity: DTX3 antibodies vary in their species reactivity profiles, with some recognizing only human DTX3 while others cross-react with mouse, rat, or other species .

The following table summarizes key specifications of commercially available DTX3 antibodies:

How should DTX3 antibody specificity be validated in experimental systems?

Rigorous validation of DTX3 antibody specificity is essential for reliable research outcomes. A comprehensive validation strategy includes:

• Positive and negative control tissues/cells: Based on the provided data, validated positive controls for Western blotting include mouse kidney, ovary, testis, and brain tissues, as well as human cell lines such as hTERT-RPE1 . For IHC, human kidney and colon tissues serve as reliable positive controls .

• Knockdown/knockout validation: The gold standard for antibody validation is demonstrating loss of signal in DTX3 knockdown or knockout samples. Use DTX3-specific shRNA constructs targeting the coding region or 3'-UTR sequence as described in previous studies .

• Molecular weight verification: The expected molecular weight for DTX3 is 38 kDa . Always verify that your detected band corresponds to this size, as non-specific binding at other molecular weights may indicate poor antibody specificity.

• Cross-reactivity assessment: DTX3 belongs to the Deltex family, which includes DTX1, DTX2, DTX3L, and DTX4. Verify that your antibody does not cross-react with these homologs by using recombinant proteins or cell lysates with differential expression of these family members .

What are the optimal conditions for Western blot detection of DTX3?

Successful Western blot detection of DTX3 requires careful optimization of several parameters:

What are the key considerations for immunohistochemical detection of DTX3 in tissue samples?

Optimal IHC protocols for DTX3 detection should address these critical factors:

• Antigen retrieval: Use TE buffer pH 9.0 for optimal antigen retrieval, although citrate buffer pH 6.0 may serve as an alternative . Heat-induced epitope retrieval (HIER) should be performed for 30 minutes in the selected buffer.

• Antibody dilution: Start with a dilution range of 1:20-1:200 and optimize based on signal-to-noise ratio . The optimal dilution may vary significantly between tissues and fixation methods.

• Detection system: An avidin-biotin complex (ABC) system such as VECTSDTSIN Elite ABC Kit has demonstrated good results in previous studies .

• Controls and counterstaining: Include positive control tissues (human kidney or colon), negative controls (primary antibody omission), and hematoxylin counterstaining for nuclear visualization .

• Evaluation parameters: When scoring DTX3 expression, consider both staining intensity and percentage of positive cells, particularly noting the subcellular localization pattern (nuclear vs. cytoplasmic predominance).

How can DTX3 antibodies be utilized to study its role in protein ubiquitination pathways?

DTX3 functions as an E3 ubiquitin ligase that promotes NOTCH2 ubiquitination and degradation through the proteasome pathway . To investigate this mechanism:

What approaches can be used to investigate DTX3's role in cancer progression?

The potential role of DTX3 as an anti-oncogene in esophageal carcinoma opens several research avenues:

• Expression correlation studies: Analyze the relationship between DTX3 and NOTCH2 expression in patient samples using IHC or Western blot. Previous studies have shown a significant negative correlation between DTX3 and NOTCH2 in esophageal cancer tissues .

• Functional studies: Utilize DTX3 overexpression or knockdown in cancer cell lines to assess effects on:

  • Proliferation (using Ki-67 or PCNA as markers)

  • Migration and invasion capabilities

  • Apoptotic pathway activation (measuring Bad, BAX expression)

  • AKT signaling pathway activity (p-AKT levels)

• In vivo tumor models: Establish xenograft models using cells with modified DTX3 expression to evaluate tumorigenicity and potential therapeutic applications.

• Biomarker potential: Investigate the prognostic significance of DTX3 expression in patient cohorts, correlating expression levels with clinical outcomes and treatment responses.

How can protein-protein interactions involving DTX3 be effectively studied?

Understanding DTX3's interaction network is crucial for elucidating its functions:

• Yeast two-hybrid screening: This approach has successfully identified DTX3 as an E3 ligase for NOTCH2 . Researchers can employ similar strategies using DTX3 as bait to identify additional interaction partners.

• Co-immunoprecipitation: Use DTX3 antibodies for immunoprecipitation followed by mass spectrometry or targeted Western blot to identify and validate interaction partners. Previous studies have shown that endogenous NOTCH2 forms a complex with DTX3 but not with other Deltex family members (DTX1, DTX2, DTX4) .

• Immunofluorescence co-localization: Combine DTX3 antibodies with antibodies against potential interaction partners to visualize co-localization in subcellular compartments. For example, NOTCH2 and DTX3 have been shown to co-localize in the cytoplasm of esophageal carcinoma cells .

• Proximity ligation assay (PLA): This technique provides higher sensitivity for detecting protein-protein interactions in situ and could be valuable for confirming interactions identified through other methods.

What are common issues encountered in Western blot detection of DTX3 and their solutions?

Researchers frequently face several challenges when detecting DTX3 by Western blot:

• Weak or absent signal: This may result from low DTX3 expression in your sample. Consider:

  • Using tissues with known high expression (kidney, ovary, testis, brain)

  • Increasing protein loading (up to 50-80 μg total protein)

  • Reducing antibody dilution (start with 1:2000)

  • Extending exposure time or using signal enhancement systems

• Multiple bands: Non-specific bands may appear due to:

  • Cross-reactivity with other Deltex family members

  • Degradation products or post-translational modifications

  • Solution: Include positive controls with known molecular weight (38 kDa for DTX3) and validate specificity using knockdown/knockout samples

• High background: If experiencing excessive background, try:

  • Increasing blocking time or concentration

  • Using more stringent washing conditions

  • Increasing antibody dilution (up to 1:10000 for WB)

  • Testing alternative blocking agents (milk vs. BSA)

What factors affect immunohistochemical staining quality for DTX3?

Optimizing IHC for DTX3 detection requires addressing several variables:

• Fixation and processing artifacts: Overfixation can mask epitopes while underfixation may lead to poor tissue morphology. Standardize fixation times and conditions across samples.

• Antigen retrieval efficiency: DTX3 detection is significantly improved with TE buffer pH 9.0, but some tissues may require optimized conditions. Compare different retrieval methods systematically .

• Antibody concentration optimization: The recommended range of 1:20-1:200 is broad . Perform a dilution series to determine optimal concentration for specific tissue types.

• Detection system sensitivity: For low abundance expression, consider amplification systems like tyramide signal amplification (TSA) in addition to standard ABC methods .

• Counterstaining interference: Heavy hematoxylin counterstaining may obscure weak cytoplasmic DTX3 signals. Adjust counterstaining intensity accordingly.

What emerging applications may benefit from DTX3 antibodies?

Several promising research directions could leverage DTX3 antibodies:

• Single-cell analysis: Adapting DTX3 antibodies for multiplexed immunofluorescence or mass cytometry could reveal cell type-specific expression patterns and functional relationships.

• Therapeutic target validation: As DTX3 emerges as a potential anti-oncogene, antibodies will be crucial for validating its role in preclinical models and eventually in clinical trials.

• Biomarker development: The negative correlation between DTX3 and NOTCH2 in esophageal cancer suggests potential diagnostic or prognostic applications that will require highly specific antibodies .

• Post-translational modification mapping: Developing modification-specific antibodies (phospho-DTX3, ubiquitinated-DTX3) could illuminate regulatory mechanisms controlling DTX3 function.

How might the DTX3-NOTCH2 axis be targeted therapeutically?

Understanding the mechanistic details of DTX3-mediated NOTCH2 regulation opens several therapeutic possibilities:

• E3 ligase activity modulation: Small molecules that enhance DTX3's E3 ligase activity could accelerate NOTCH2 degradation, potentially suppressing tumor growth in cancers with elevated NOTCH2.

• Protein-protein interaction targeting: Compounds that stabilize the DTX3-NOTCH2 interaction could enhance NOTCH2 degradation.

• Gene therapy approaches: Viral vector-mediated DTX3 overexpression could be explored in tumors with low DTX3 and high NOTCH2 expression .

• Combination therapies: DTX3-targeting approaches could potentially sensitize tumors to conventional therapies by modulating NOTCH2-dependent survival pathways.