HECTD3 Antibody

What is HECTD3 and why is it significant in cellular research?

HECTD3 (HECT domain-containing protein 3) is an E3 ubiquitin ligase that plays crucial roles in several biological processes through its ability to mediate non-degradative ubiquitination of target proteins. HECTD3 has emerged as a significant regulator in diverse pathophysiological contexts, including viral infections, autoimmune disorders, and cancer progression .

The significance of HECTD3 lies in its ability to modify proteins through specific types of ubiquitin linkages (such as K27, K29, and K33) that often don't lead to degradation but instead alter protein function, localization, or interactions . This non-proteolytic ubiquitination represents an important regulatory mechanism in cell signaling pathways.

What are the recommended applications for HECTD3 antibodies in experimental research?

Based on validated data, HECTD3 antibodies (such as 11487-1-AP) have been successfully employed in multiple applications:

It's important to note that each experimental system may require optimization of antibody concentration to achieve optimal signal-to-noise ratios .

How should sample preparation be optimized when using HECTD3 antibodies?

For optimal results with HECTD3 antibodies, sample preparation should follow these methodological guidelines:

• For Western blot: Lyse cells in RIPA buffer supplemented with protease inhibitors and phosphatase inhibitors (if studying phosphorylation events). Denaturation at 95°C for 5 minutes in reducing sample buffer is generally effective for HECTD3 detection.

• For immunoprecipitation: Gentler lysis conditions using NP-40 or Triton X-100 based buffers (typically 0.5-1%) help preserve protein-protein interactions. When studying HECTD3's E3 ligase activity, consider including deubiquitinase inhibitors (like N-ethylmaleimide) in lysis buffers to preserve ubiquitination status.

• For immunohistochemistry: Formalin-fixed paraffin-embedded tissues generally work well, with antigen retrieval using TE buffer at pH 9.0 showing better results than citrate buffer (pH 6.0) for HECTD3 detection in tissues like human stomach .

• Post-translational modification studies: When analyzing HECTD3-mediated ubiquitination, consider using specialized approaches like ubiquitin-remnant profiling or tandem ubiquitin binding entity (TUBE) pulldowns to enrich for ubiquitinated proteins.

How should knockout/knockdown validation be designed when studying HECTD3 function?

When establishing HECTD3 knockout or knockdown models, a comprehensive validation approach should include:

• mRNA expression verification: Use qRT-PCR with primers targeting different exons of HECTD3 to confirm transcript reduction.

• Protein expression verification: Western blot analysis using validated HECTD3 antibodies like 11487-1-AP at 1:1000-1:3000 dilution. Multiple HECTD3 antibodies targeting different epitopes can provide more robust validation .

• Functional validation: Since HECTD3 is an E3 ligase, assess changes in ubiquitination patterns of known substrates such as PKR, Malt1, Stat3, c-MYC, or TRIOBP . This typically involves immunoprecipitation of the substrate followed by ubiquitin blotting.

• Phenotypic rescue experiments: To confirm specificity, perform rescue experiments by reintroducing wild-type HECTD3 or catalytically inactive mutants (typically mutations in the HECT domain). For example, research has demonstrated that reintroduction of HECTD3 in knockdown gastric cancer cells rescued proliferation phenotypes .

• Controls: Include appropriate controls, such as non-targeting shRNA/siRNA or scrambled guide RNA for CRISPR/Cas9 approaches.

What are the key considerations for studying HECTD3-mediated ubiquitination in experimental settings?

When investigating HECTD3-mediated ubiquitination:

• Linkage-specific analysis: HECTD3 has been shown to mediate specific ubiquitin linkages including K27, K29, and K33-linked polyubiquitination . Use linkage-specific ubiquitin antibodies or mass spectrometry approaches to identify the precise ubiquitin chain topology.

• Domain mapping: To determine which regions of HECTD3 interact with substrates, construct domain deletion mutants. Research has shown that the DOC domain (amino acids 215-393) of HECTD3 interacts with the CP and bHLHZ domains of c-MYC .

• Stability assessment: Although HECTD3-mediated ubiquitination often doesn't lead to degradation, protein stability should still be assessed. Cycloheximide chase assays have shown that HECTD3 overexpression slows the degradation rate of c-MYC .

• Control for non-specific effects: Include catalytically inactive HECTD3 mutants (typically with mutations in the HECT domain) as controls to distinguish between scaffolding and catalytic functions.

• Deubiquitinase inhibition: When studying ubiquitination, include deubiquitinase inhibitors such as PR-619 (broad-spectrum) or more specific inhibitors in lysis buffers to preserve ubiquitin modifications.

How do sample source and preparation affect HECTD3 antibody performance?

Sample source and preparation significantly impact HECTD3 antibody performance:

• Cell/tissue-specific expression: HECTD3 expression varies across tissues and cell types. While the 11487-1-AP antibody has been validated in MCF-7, MDA-MB-453s cells (human) and various mouse cells, expression levels may vary in other systems .

• Species considerations: The HECTD3 antibody 11487-1-AP has been validated for human and mouse samples . For other species, sequence homology analysis and preliminary validation are recommended.

• Subcellular fractionation: HECTD3 has been observed to interact with different substrates depending on cellular compartments and stimulation conditions. For instance, HECTD3-PKR interaction is enhanced by RNA viruses and cytosolic RNA analogues but not by DNA viruses or TLR ligands . Consider subcellular fractionation when studying context-specific interactions.

• Fixation effects: For IHC and IF applications, fixation conditions affect epitope accessibility. Antigen retrieval with TE buffer at pH 9.0 has shown better results than citrate buffer (pH 6.0) for HECTD3 detection in human stomach tissue .

How can HECTD3 antibodies be utilized to investigate viral infection mechanisms?

HECTD3 antibodies can be strategically employed to investigate viral infection mechanisms based on recent findings that HECTD3 promotes RNA virus replication while inhibiting anti-viral immune responses:

• Infection-induced changes in HECTD3: Western blot analysis using HECTD3 antibodies at 1:1000 dilution can track changes in HECTD3 expression following viral infection. Research has shown that VSV infection downregulates HECTD3 at the transcriptional level rather than through proteasome-mediated degradation .

• HECTD3-PKR interaction studies: Co-immunoprecipitation assays can be used to investigate how RNA viruses enhance the interaction between HECTD3 and PKR. This interaction has been shown to be specifically enhanced by RNA viruses and cytosolic RNA analogues but not by DNA viruses or TLR ligands .

• Ubiquitination analysis: HECTD3 mediates K33-linked polyubiquitination of PKR, disrupting PKR dimerization and phosphorylation while promoting PKR-IKK complex formation. Using HECTD3 antibodies in combination with ubiquitin linkage-specific antibodies can help map these dynamics during viral infection .

• Viral replication assessment: HECTD3 has differential effects on RNA versus DNA viruses. In HECTD3-deficient cells, RNA virus (VSV, SeV) replication is inhibited while DNA virus (HSV-1) replication is enhanced. Combining HECTD3 antibody staining with viral protein detection can help visualize these effects .

What methodologies are recommended for investigating HECTD3's role in cancer progression?

To investigate HECTD3's role in cancer progression, consider these methodological approaches:

• Expression analysis in cancer tissues: IHC using HECTD3 antibody (1:200-1:800 dilution) can assess HECTD3 expression in tumor versus normal tissues. This is particularly relevant for gastric cancer where HECTD3 has been implicated in tumor progression .

• Proliferation and cell cycle studies: After HECTD3 knockdown or overexpression, measure:

  • Cell proliferation using MTT assays

  • DNA synthesis through BrdU incorporation

  • Clone formation capabilities via soft-agar assays

  • Cell cycle distribution by flow cytometry

  Research has shown that HECTD3 knockdown in gastric cancer cells arrests the cell cycle in G1 phase and induces apoptosis .

• Signaling pathway analysis: Western blotting can assess how HECTD3 manipulation affects expression of c-MYC, CDK2, CDK4, and p21. HECTD3 knockdown reduces c-MYC, CDK2, and CDK4 while increasing p21 expression in gastric cancer cells .

• c-MYC ubiquitination studies: Since HECTD3 mediates K29-linked polyubiquitination of c-MYC, immunoprecipitation of c-MYC followed by ubiquitin blotting can reveal how HECTD3 regulates this key oncogenic transcription factor .

• Domain interaction mapping: Co-immunoprecipitation studies using domain-specific constructs have shown that the DOC domain of HECTD3 interacts with the CP and bHLHZ domains of c-MYC, providing insights into the structural basis of HECTD3's oncogenic functions .

How can HECTD3 antibodies be used to investigate autoimmune disease mechanisms?

HECTD3 antibodies can be instrumental in investigating autoimmune disease mechanisms, particularly in the context of experimental autoimmune encephalomyelitis (EAE) and Th17-mediated pathology:

• T cell differentiation studies: Western blot analysis using HECTD3 antibodies can track changes in HECTD3 expression during T helper cell differentiation. HECTD3 has been shown to promote pathogenic Th17 cell development in EAE .

• Protein interaction networks: Co-immunoprecipitation with HECTD3 antibodies can identify interaction partners in T cells during autoimmune conditions. Research has demonstrated that HECTD3 associates with Malt1 in CD4+ T cells during EAE .

• Ubiquitination analysis in immune cells: HECTD3 mediates non-degradative polyubiquitination of Malt1 (K27 and K29-linked) and Stat3 (K27-linked) in T cells. Immunoprecipitation of these targets followed by ubiquitin blotting can reveal how HECTD3 regulates these key immune signaling molecules .

• In vivo disease models: HECTD3 knockout mice show attenuated EAE severity and reduced immune cell infiltration in the CNS. Immunohistochemistry using antibodies against HECTD3 and immune cell markers can help visualize these effects in tissue sections .

• Signaling pathway analysis: Since HECTD3 affects Stat3 phosphorylation at tyrosine-705, phospho-specific antibodies can be used alongside HECTD3 antibodies to track how HECTD3 manipulations affect Th17-related signal transduction .

What are common technical challenges when using HECTD3 antibodies and how can they be addressed?

When working with HECTD3 antibodies, researchers may encounter several technical challenges:

• High background in Western blots:

  • Increase blocking time (5% non-fat milk or BSA for 2 hours)

  • Optimize antibody dilution (start with 1:1000 and adjust as needed)

  • Include Tween-20 (0.1%) in wash buffers

  • Consider using different blocking agents (BSA vs. milk)

• Weak or no signal in immunoprecipitation:

  • Increase antibody amount (up to 4 μg for 3 mg of lysate)

  • Extend incubation time (overnight at 4°C)

  • Ensure HECTD3 is expressed in your cell type (check literature or databases)

  • Verify extraction conditions preserve protein-protein interactions

  • Consider crosslinking approaches for transient interactions

• Inconsistent results in immunohistochemistry:

  • Optimize antigen retrieval (TE buffer pH 9.0 is recommended over citrate buffer)

  • Adjust antibody concentration (1:200-1:800 dilution range)

  • Ensure proper fixation protocols (overfixation can mask epitopes)

  • Include positive control tissues (human stomach tissue shows positive staining)

• Non-specific bands in Western blot:

  • Use HECTD3 knockout/knockdown samples as negative controls

  • Increase washing stringency

  • Optimize primary antibody concentration

  • Consider using gradient gels for better separation around the expected 97 kDa band

How should researchers interpret contradictory findings in HECTD3 functional studies?

When encountering contradictory findings in HECTD3 studies, consider these analytical approaches:

• Context-specific effects: HECTD3 exhibits distinct effects depending on cellular context. For example, HECTD3 promotes RNA virus replication but inhibits DNA virus replication . Similarly, its effects may differ between immune cells and cancer cells. Carefully document cell types and stimulation conditions.

• Substrate specificity: HECTD3 targets different substrates in different contexts (PKR in viral infection , Malt1/Stat3 in autoimmunity , c-MYC in cancer ). Contradictory findings might reflect different substrate availability or activation.

• Ubiquitin linkage analysis: HECTD3 can mediate different ubiquitin linkages (K27, K29, K33) with distinct functional outcomes. Use linkage-specific antibodies or mass spectrometry to determine the exact modification.

• Technical considerations:

  • Antibody specificity (validate with knockout/knockdown controls)

  • Expression level artifacts (overexpression vs. endogenous studies)

  • Genetic background effects (particularly in mouse models)

  • Temporal dynamics (acute vs. chronic effects)

• Integration with pathway analysis: Since HECTD3 affects multiple signaling pathways, contradictory findings might reflect different pathway states. Use pathway inhibitors or activators to dissect these interactions.

How can researchers differentiate between direct and indirect effects of HECTD3 in experimental systems?

Distinguishing direct from indirect effects of HECTD3 requires specific experimental approaches:

• In vitro ubiquitination assays: Reconstitute HECTD3-mediated ubiquitination with purified components (E1, E2, HECTD3, substrate, and ubiquitin). Direct substrates will be ubiquitinated in this minimal system.

• Structure-function analysis: Create domain mutants of HECTD3 to map interaction surfaces. Research has shown that the DOC domain (215-393aa) of HECTD3 interacts with c-MYC , while other domains may interact with different substrates.

• Catalytically inactive mutants: Compare effects of wild-type HECTD3 with catalytically inactive mutants (typically in the HECT domain). Differences indicate E3 ligase activity-dependent (direct) effects, while shared effects suggest scaffolding (potentially indirect) functions.

• Proximity labeling approaches: BioID or APEX2 fused to HECTD3 can identify proteins in close proximity, helping distinguish direct from indirect interactors.

• Temporal analysis: Direct effects typically occur more rapidly than indirect ones. Time-course experiments following HECTD3 induction or inhibition can help distinguish these kinetics.

• Competitive binding assays: For suspected direct interactions, use peptide competition or mutant substrates to block specific binding sites and verify direct substrate recognition.

What emerging techniques might enhance HECTD3 antibody applications in cutting-edge research?

Several emerging techniques show promise for expanding HECTD3 antibody applications:

• Proximity ligation assays (PLA): This technique can visualize HECTD3 interactions with substrate proteins with high sensitivity and spatial resolution within cells. PLA could be particularly valuable for detecting context-specific interactions between HECTD3 and targets like PKR during viral infection or c-MYC in cancer cells.

• CRISPR epitope tagging: Endogenous tagging of HECTD3 using CRISPR/Cas9 allows visualization and pulldown of HECTD3 at physiological expression levels, avoiding overexpression artifacts that may complicate traditional antibody applications.

• Ubiquitin chain-specific sensors: Linkage-specific ubiquitin binding domains fused to fluorescent proteins can track specific ubiquitin modifications (K27, K29, K33) mediated by HECTD3 in live cells.

• Single-cell applications: Adapting HECTD3 antibodies for single-cell proteomics or CyTOF could reveal cell-to-cell variability in HECTD3 expression and function during immune responses or in heterogeneous tumor samples.

• Spatial transcriptomics/proteomics integration: Combining HECTD3 antibody staining with spatial transcriptomics could correlate HECTD3 protein expression with gene expression programs in tissues, providing insights into its functions in complex tissues.

How might combining HECTD3 antibodies with other molecular tools advance understanding of ubiquitination networks?

Integrative approaches combining HECTD3 antibodies with other molecular tools can provide deeper insights into ubiquitination networks:

• Ubiquitin remnant profiling with HECTD3 manipulation: Combining HECTD3 knockdown/overexpression with ubiquitin remnant profiling (K-ε-GG peptide enrichment) and mass spectrometry can identify the global landscape of HECTD3-dependent ubiquitination sites.

• TUBE (Tandem Ubiquitin Binding Entity) pulldowns: Using linkage-specific TUBEs alongside HECTD3 antibodies can enrich for specific ubiquitin chain topologies (K27, K29, K33) mediated by HECTD3 for subsequent analysis.

• Interactome mapping: BioID or APEX2 proximity labeling fused to HECTD3 can map its dynamic interactome under different conditions, such as viral infection or cancer, revealing context-specific functions.

• Optical tools for studying HECTD3 dynamics: Optogenetic control of HECTD3 activity combined with biosensors for downstream signaling (e.g., PKR activity, NF-κB activation) could reveal the kinetics and spatial organization of HECTD3-dependent signaling.

• HECTD3 substrate prediction algorithms: Developing computational tools to predict HECTD3 substrates based on known targets could generate hypotheses for novel functions, with antibody-based validation of predicted interactions.

What are the implications of HECTD3 research for therapeutic development?

Current research highlights several therapeutic implications where HECTD3 antibodies could play crucial roles in drug development:

• Antiviral therapy potential: Since HECTD3 deficiency restricts RNA virus replication and reduces inflammatory responses in vitro and in vivo, HECTD3 inhibitors might serve as dual-action antivirals that both limit viral replication and dampen harmful inflammation . Antibodies could be used to screen for compounds disrupting HECTD3-PKR interaction.

• Autoimmune disease applications: HECTD3 knockout mice show reduced severity of experimental autoimmune encephalomyelitis, suggesting HECTD3 inhibition could be beneficial in multiple sclerosis and other Th17-driven autoimmune diseases . HECTD3 antibodies could help identify specific interactions to target.

• Cancer therapy development: HECTD3 knockdown inhibits gastric cancer cell proliferation, induces cell cycle arrest, and promotes apoptosis . HECTD3 antibodies can facilitate screening for inhibitors that might show selective activity against HECTD3-dependent cancers.

• Biomarker development: HECTD3 antibodies in IHC could help assess HECTD3 expression in patient samples, potentially stratifying patients for targeted therapies or providing prognostic information.

• Target validation approaches: For therapeutic development, combining HECTD3 antibodies with conditional knockout models and specific inhibitors can provide multi-level validation of HECTD3 as a drug target and help distinguish on-target from off-target drug effects.