HEC3 Antibody

What is HECTD3 and why is it important to study?

HECTD3 is an E3 ubiquitin ligase that catalyzes the addition of ubiquitin to specific substrate proteins, targeting them for various cellular fates. It contains a DOC domain (amino acids 216-393) that is responsible for substrate recruitment and a HECT domain (amino acids 512-861) that catalyzes the ubiquitination reaction . HECTD3 has been implicated in multiple critical cellular processes including:

• Inflammation response regulation through IKKα stabilization and nuclear localization

• Cancer progression, particularly in gastric cancer, by mediating the polyubiquitination of c-MYC

• Metastasis through modulation of adhesion molecule expression in endothelial cells

Understanding HECTD3 function is essential for elucidating these pathways and potentially developing therapeutic strategies for diseases where HECTD3 activity is dysregulated.

What types of experiments can be performed using HECTD3 antibody?

HECTD3 antibody can be utilized in multiple experimental approaches:

• Western Blot (WB): For detecting and quantifying HECTD3 protein expression in cell lysates and tissue samples. The recommended dilution is 1:1000-1:3000 .

• Immunoprecipitation (IP): For isolating HECTD3 and its interacting proteins from complex biological samples. This application is particularly useful for studying protein-protein interactions. Use 0.5-4.0 μg of antibody for 1.0-3.0 mg of total protein lysate .

• Immunohistochemistry (IHC): For visualizing HECTD3 localization in tissue sections, with recommended dilution of 1:200-1:800. Antigen retrieval with TE buffer pH 9.0 is suggested, although citrate buffer pH 6.0 may also be used .

• Co-immunoprecipitation (Co-IP): For investigating protein interactions between HECTD3 and potential substrates like IKKα or c-MYC .

• Chromatin Immunoprecipitation (ChIP): While not directly using HECTD3 antibody, researchers have used IKKα antibody in ChIP assays to study how HECTD3 affects IKKα recruitment to promoter regions of genes like E-selectin, ICAM-1, and VCAM-1 .

How should HECTD3 antibody be validated before experimental use?

Proper validation of HECTD3 antibody is crucial for obtaining reliable results:

• Specificity testing: Verify antibody specificity through:

  • Western blot analysis in cell lines known to express HECTD3 (e.g., MCF-7, MDA-MB-453s)

  • Comparison with negative controls (HECTD3 knockout or knockdown samples)

  • Testing multiple antibodies targeting different epitopes of HECTD3

• Application-specific validation:

  • For WB: Confirm the antibody detects a band of the expected molecular weight

  • For IP: Verify successful pulldown of HECTD3 through subsequent western blot

  • For IHC: Compare staining patterns with mRNA expression data and include appropriate positive and negative controls

• Cross-reactivity assessment: Test the antibody on samples from different species if cross-species reactivity is claimed or needed.

How can HECTD3 antibody be used to investigate HECTD3's role in ubiquitination pathways?

HECTD3 mediates specific types of polyubiquitination, particularly K27-linked and K63-linked polyubiquitination . To investigate these pathways:

• Ubiquitination assay setup:

  • Transfect cells with expression vectors for the substrate of interest (e.g., IKKα or c-MYC), HECTD3, and HA-tagged ubiquitin

  • Treat cells with proteasome inhibitors (e.g., MG132) to prevent degradation of ubiquitinated proteins

  • Lyse cells under denaturing conditions to disrupt non-covalent protein interactions

  • Immunoprecipitate the substrate using a specific antibody

  • Detect ubiquitination by western blotting using anti-HA antibody

• Linkage-specific ubiquitination analysis:

  • Use linkage-specific anti-ubiquitin antibodies (K27, K48, K63) to determine the type of polyubiquitin chains

  • Alternatively, employ ubiquitin mutants where only one lysine is available (K-only mutants) to identify the linkage type

• Mapping ubiquitination sites:

  • Generate point mutations in potential ubiquitination sites of the substrate

  • Compare ubiquitination levels between wild-type and mutant proteins

  • For example, K296R mutation in IKKα abolished HECTD3-mediated ubiquitination

This methodological approach revealed that HECTD3 mediates K29-linked polyubiquitination of c-MYC in gastric cancer and K27/K63-linked polyubiquitination of IKKα in endothelial cells .

What are the best approaches for studying HECTD3-substrate interactions using antibody-based methods?

To effectively investigate interactions between HECTD3 and its substrates:

• Domain mapping strategy:

  • Generate truncated mutants of both HECTD3 and the potential substrate

  • Perform co-IP experiments with these mutants to identify interaction domains

  • Research has shown that the DOC domain of HECTD3 (amino acids 216-393) interacts with the SDD domain of IKKα (amino acids 408-665) and with the CP (145-320aa) and bHLHZ (321-454aa) domains of c-MYC

• Endogenous interaction confirmation:

  • Use HECTD3 antibody to immunoprecipitate endogenous HECTD3 and blot for potential substrates

  • Conversely, use substrate-specific antibodies to immunoprecipitate and blot for HECTD3

  • This approach confirmed endogenous interaction between HECTD3 and IKKα in HUVECs

• Co-localization studies:

  • Perform immunofluorescence staining using HECTD3 antibody and antibodies against potential substrates

  • Analyze co-localization using confocal microscopy

  • This method demonstrated co-localization of HECTD3 and IKKα in HUVECs

• GST-pulldown assays:

  • Express GST-tagged domains of HECTD3 or its potential substrates

  • Perform pulldown experiments with cell lysates or recombinant proteins

  • Analyze interactions by western blotting

  • This approach helped map the interaction between HECTD3's DOC domain and IKKα's SDD domain

How can HECTD3 antibody be used to investigate differences in protein stability and half-life?

HECTD3-mediated ubiquitination affects protein stability and half-life of its substrates. To investigate this:

• Cycloheximide chase assay:

  • Treat cells with cycloheximide to inhibit new protein synthesis

  • Collect samples at different time points (0, 2, 4, 8 hours)

  • Analyze protein levels by western blotting using substrate-specific antibodies

  • Compare protein half-lives between conditions (e.g., with/without HECTD3, wild-type vs. ubiquitination site mutants)

  • This approach revealed that the K296R mutant of IKKα exhibited a shorter protein half-life compared to wild-type IKKα

• Proteasome inhibition studies:

  • Treat cells with proteasome inhibitors (e.g., MG132)

  • Compare protein levels with/without inhibitor treatment

  • This can help determine if HECTD3-mediated ubiquitination leads to proteasomal degradation

• Pulse-chase analysis:

  • Label newly synthesized proteins with radioactive amino acids (pulse)

  • Chase with non-radioactive medium for various time periods

  • Immunoprecipitate the protein of interest

  • Measure radioactivity to determine protein half-life

  • Compare between conditions with normal or altered HECTD3 expression

How can HECTD3 antibody be used to study cancer progression mechanisms?

HECTD3 has been implicated in cancer progression, particularly in gastric cancer. Researchers can use HECTD3 antibody to:

• Evaluate HECTD3 expression in cancer tissues:

  • Perform IHC on cancer tissues and paired normal tissues

  • Quantify expression differences and correlate with clinical parameters

  • HECTD3 antibody has been successfully used for IHC in human stomach tissue

• Investigate HECTD3-mediated oncogenic pathways:

  • Study HECTD3 interaction with c-MYC using co-IP with HECTD3 antibody

  • Examine how HECTD3 affects c-MYC stability and activity through ubiquitination

  • Research has shown that HECTD3 mediates K29-linked polyubiquitination of c-MYC, promoting gastric cancer progression

• Analyze downstream signaling effects:

  • Use HECTD3 antibody in conjunction with antibodies against downstream signaling molecules

  • Compare signaling pathway activation in cells with normal vs. altered HECTD3 expression

  • Investigate how HECTD3 knockdown or overexpression affects cancer cell phenotypes

• Develop tissue microarrays (TMAs):

  • Create TMAs containing multiple cancer samples

  • Perform high-throughput IHC analysis using HECTD3 antibody

  • Correlate HECTD3 expression with cancer subtypes and patient outcomes

What methods can be used to study HECTD3's role in inflammation-related metastasis?

HECTD3 plays a significant role in inflammation-related metastasis by regulating adhesion molecule expression. Researchers can:

• Study HECTD3-IKKα axis in endothelial cells:

  • Use HECTD3 antibody to immunoprecipitate HECTD3 and analyze its interaction with IKKα

  • Perform nuclear/cytoplasmic fractionation and use HECTD3 antibody to track HECTD3 localization

  • Examine how HECTD3 knockdown affects IKKα nuclear translocation and stability

• Analyze adhesion molecule expression:

  • Use HECTD3 antibody alongside antibodies against E-selectin, ICAM-1, and VCAM-1

  • Compare expression levels in control and HECTD3-modified endothelial cells

  • HECTD3 has been shown to promote adhesion molecule expression in endothelial cells under inflammatory conditions

• Perform ChIP assays:

  • Use IKKα antibody for ChIP to analyze recruitment to adhesion molecule gene promoters

  • Compare IKKα recruitment between control and HECTD3-knockdown cells

  • This approach revealed that HECTD3 promotes IKKα recruitment to E-selectin, ICAM-1, and VCAM-1 promoters

• Setup in vivo metastasis models:

  • Compare metastasis in wild-type and HECTD3 knockout mice

  • Use HECTD3 antibody for IHC to analyze expression in metastatic sites

  • Examine how HECTD3 affects tumor cell adhesion in the vasculature under inflammatory conditions

What are the key considerations for optimizing HECTD3 antibody use in immunohistochemistry?

For optimal IHC results with HECTD3 antibody:

• Antigen retrieval optimization:

  • HECTD3 antibody works best with TE buffer pH 9.0 for antigen retrieval

  • Alternatively, citrate buffer pH 6.0 can be used

  • Optimize retrieval time and temperature for your specific tissue type

• Antibody dilution titration:

  • Test a range of dilutions (e.g., 1:200, 1:400, 1:800) to find optimal signal-to-noise ratio

  • Include positive control tissues known to express HECTD3 (e.g., human stomach tissue)

  • Include negative controls (primary antibody omission, isotype control)

• Detection system selection:

  • Choose appropriate detection systems based on sensitivity requirements

  • For low expression, consider using amplification systems like tyramide signal amplification

  • For co-localization studies, select fluorescent secondary antibodies with minimal spectral overlap

• Sample preparation considerations:

  • Fixation type and duration can affect epitope availability

  • Section thickness (typically 4-5 μm) affects antibody penetration

  • Fresh vs. archived tissue samples may require different protocols

How can researchers troubleshoot common issues with HECTD3 antibody in western blotting?

Common western blotting issues and solutions:

• Weak or no signal:

  • Increase antibody concentration (try 1:1000 before moving to 1:500 or higher)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Ensure sufficient protein loading (at least 20-30 μg total protein)

  • Check protein transfer efficiency with reversible staining

  • Verify HECTD3 expression in your cell line/tissue (MCF-7 and MDA-MB-453s are known to express HECTD3)

• High background:

  • Dilute antibody further (try 1:3000)

  • Increase blocking time or concentration

  • Use more stringent washing conditions

  • Try different blocking agents (milk vs. BSA)

  • Consider using more specific secondary antibodies

• Multiple bands:

  • Verify expected molecular weight of HECTD3

  • Additional bands may represent degradation products, post-translational modifications, or splice variants

  • Increase sample denaturation time/temperature

  • Add protease inhibitors during sample preparation

• Inconsistent results:

  • Standardize protein extraction methods

  • Use internal loading controls (β-actin, GAPDH)

  • Prepare fresh antibody dilutions for each experiment

  • Consider lot-to-lot variations in antibody performance

What controls should be included when using HECTD3 antibody in research experiments?

Proper controls are essential for reliable results:

• Positive controls:

  • Cell lines known to express HECTD3 (MCF-7, MDA-MB-453s)

  • Tissues with confirmed HECTD3 expression (human stomach tissue)

  • Overexpression systems with tagged HECTD3

• Negative controls:

  • HECTD3 knockout or knockdown samples

  • Cell lines with naturally low HECTD3 expression

  • Primary antibody omission controls

  • Isotype controls (for flow cytometry or IHC)

• Specificity controls:

  • Peptide competition assay (pre-incubating antibody with excess immunizing peptide)

  • Testing multiple antibodies targeting different HECTD3 epitopes

  • Validation in HECTD3 knockout models

• Processing controls:

  • For ubiquitination studies: include proteasome inhibitors

  • For protein interaction studies: include cross-linking controls

  • For ChIP assays: include input controls and IgG controls

How can researchers distinguish between different types of ubiquitin linkages mediated by HECTD3?

HECTD3 has been shown to catalyze different types of ubiquitin linkages depending on the substrate. To distinguish between them:

• Use linkage-specific ubiquitin antibodies:

  • Commercial antibodies specific for K27-, K48-, or K63-linked polyubiquitin chains

  • Immunoblot analysis of immunoprecipitated substrates

  • This approach revealed that HECTD3 mediates K27- and K63-linked polyubiquitination of IKKα

• Employ ubiquitin mutants:

  • Utilize ubiquitin mutants where all lysines except one are mutated to arginine (K-only mutants)

  • Compare ubiquitination patterns with different K-only mutants

  • Research showed that K27- and K63-only ubiquitin supported HECTD3-catalyzed IKKα polyubiquitination

• Mass spectrometry analysis:

  • Immunoprecipitate ubiquitinated proteins

  • Perform tryptic digestion and analyze by mass spectrometry

  • Identify ubiquitin remnant signatures that indicate specific linkage types

• Functional validation:

  • Generate substrate mutants that cannot be ubiquitinated at specific sites

  • Compare cellular phenotypes between wild-type and mutant proteins

  • For example, K296R mutation in IKKα prevented HECTD3-mediated stabilization

What methods can researchers use to investigate the dual nuclear and cytoplasmic functions of HECTD3?

HECTD3 appears to have both cytoplasmic and nuclear functions. To investigate this:

• Nuclear/cytoplasmic fractionation:

  • Separate nuclear and cytoplasmic fractions from cells

  • Analyze HECTD3 distribution by western blotting

  • Compare distribution under different conditions (e.g., inflammatory stimulation)

  • This approach showed that HECTD3 knockdown decreased nuclear localization of IKKα in LPS-stimulated HUVECs

• Immunofluorescence co-localization:

  • Perform immunofluorescence staining using HECTD3 antibody

  • Co-stain with nuclear markers (e.g., DAPI) and specific interaction partners

  • Analyze subcellular distribution using confocal microscopy

  • This method demonstrated colocalization of HECTD3 with IKKα in HUVECs

• Chromatin association studies:

  • Perform biochemical fractionation to isolate chromatin-bound proteins

  • Analyze HECTD3 association with chromatin by western blotting

  • Use ChIP assays to investigate HECTD3 recruitment to specific genomic loci

• Nuclear export/import inhibition:

  • Treat cells with nuclear export inhibitors (e.g., leptomycin B)

  • Analyze changes in HECTD3 distribution and function

  • Use this approach to determine if nuclear-cytoplasmic shuttling is important for HECTD3 function

What emerging applications of HECTD3 antibody are being developed for research?

Emerging applications include:

• Single-cell analysis:

  • Using HECTD3 antibody for single-cell western blotting

  • Application in mass cytometry (CyTOF) for high-dimensional analysis

  • Integration with spatial transcriptomics for correlating protein and mRNA expression

• Proximity labeling approaches:

  • Combining HECTD3 antibody with proximity ligation assays to visualize protein interactions in situ

  • Development of HECTD3-BioID or APEX2 fusion proteins for proximity-dependent biotinylation

  • These approaches could identify novel HECTD3 interactors beyond known substrates like IKKα and c-MYC

• Live-cell imaging:

  • Development of intrabodies based on HECTD3 antibody sequences

  • Application in tracking HECTD3 dynamics during cellular processes

  • FRET-based sensors to monitor HECTD3-substrate interactions in real-time

• Therapeutic targeting:

  • Using HECTD3 antibody to validate HECTD3 as a therapeutic target

  • Development of antibody-based inhibitors of HECTD3-substrate interactions

  • Potential applications in cancer therapy based on HECTD3's role in gastric cancer progression