HECTD1 Antibody

What are the validated applications for HECTD1 antibodies?

HECTD1 antibodies have been successfully tested in multiple applications including:

For optimal results, antibody dilutions should be empirically determined for each experimental system as sensitivity may vary based on protein expression levels and sample preparation methods.

How should samples be prepared for HECTD1 detection in immunostaining?

For immunofluorescence detection of HECTD1:

• Seed cells into 24-well plates containing 12-mm round cover glass slips

• Prior to treatment, serum-starve cells for 16 hours

• Fix cells with 4% formaldehyde for 15 minutes at room temperature

• Permeabilize with 0.15% Triton-X100/PBS for 15 minutes

• Block with 5% BSA/PBS for 1 hour at room temperature

• Incubate with anti-HECTD1 primary antibody (1:200 dilution) overnight at 4°C

• Wash with PBS (3 times, 15 minutes each)

• Incubate with Alexa Fluor-conjugated secondary antibodies (1:500) for 1 hour

• Stain nuclei with DAPI for 3 minutes

• Mount with anti-fade mountant and visualize using confocal microscopy

For IHC applications, antigen retrieval with TE buffer pH 9.0 is recommended, though citrate buffer pH 6.0 can be used as an alternative .

Which tissues and cell lines reliably express HECTD1?

HECTD1 is broadly expressed across multiple tissue types:

HECTD1 expresses broadly in nervous system, cardiovascular tissues, and epithelial cells, making these tissues suitable for positive controls in antibody validation studies .

How can I validate specificity of HECTD1 antibodies in knockout/knockdown systems?

A multi-step validation approach is recommended:

• Cell line selection: Utilize established cell models such as HEK293T, HeLa, or TF-1 cells that reliably express HECTD1

• Knockout/knockdown strategies:

  • Generate stable HECTD1 knockdown clones using validated shRNAs targeting HECTD1 (e.g., those corresponding to catalog numbers FI316529, FI316530, FI316531, and F1316532)

  • Use CRISPR/Cas9 to generate HECTD1 knockout cell lines as demonstrated in HEK293T cells

• Validation experiments:

  • Western blot analysis comparing wild-type and knockout/knockdown samples

  • Include antibody specificity controls such as the HECTD1 XC mouse line where a gene trap insertion disrupts the C-terminal domain, allowing for negative control validation

  • For transfected samples, include both wild-type HECTD1 and ligase-deficient HECTD1 C2579G constructs

Researchers should note that knockout validation provides more definitive evidence of antibody specificity than knockdown approaches, as residual protein can complicate interpretation in knockdown systems .

What are the optimal protocols for immunoprecipitation of HECTD1 and its interaction partners?

For co-immunoprecipitation of endogenous HECTD1 and its interaction partners:

Protocol for stringent conditions (endogenous proteins):

• Prepare tissue/cell lysates in RIPA buffer

• Incubate lysate with anti-HECTD1 antibody overnight at 4°C

• Add protein A/G beads and incubate for 2-4 hours

• Wash beads 4-5 times with RIPA buffer

• Elute bound proteins with SDS sample buffer and analyze by western blotting

Protocol for overexpressed proteins:

• Transfect cells with tagged HECTD1 constructs (e.g., HA-HECTD1) and potential interacting partners (e.g., Myc-RARA)

• Lyse cells in appropriate buffer (less stringent than RIPA)

• Immunoprecipitate using anti-tag antibodies (e.g., anti-HA agarose beads)

• Perform specific elution with HA peptide to minimize non-specific binding

• Verify elution efficiency with glycine elution as a secondary step

• Analyze eluates by western blotting or mass spectrometry

When studying HECTD1 interactions, it's critical to include appropriate controls:

• IgG control antibody

• Ligase-deficient HECTD1 mutant (C2579G) to distinguish between substrate binding and catalytic activity

• Empty vector controls for tagged protein experiments

How can I design experiments to study HECTD1's role in substrate ubiquitination?

To analyze HECTD1-mediated ubiquitination of specific substrates:

• Experimental design approach:

  • Co-transfect cells with Myc-tagged substrate (e.g., Myc-RARA), HA-HECTD1 (wild-type), and ligase-deficient HECTD1 C2579G

  • Treat cells with proteasome inhibitor (e.g., ALLN) 4 hours before lysis to prevent degradation of ubiquitinated proteins

  • Immunoprecipitate substrate with anti-Myc antibody

  • Detect ubiquitination by western blotting with anti-ubiquitin antibodies

• Ubiquitin chain-specific detection:

  • Use antibodies that distinguish between different ubiquitin linkages:

    • FK1 antibody: detects only polyubiquitinated proteins

    • FK2 antibody: detects both mono- and polyubiquitinated proteins

  • Consider that relative affinity for diverse polyubiquitin chains differs between FK1 and FK2 antibodies

• Controls for ubiquitination assays:

  • Ligase-deficient HECTD1 (C2579G) acts as a dominant negative

  • Include HECTD1 knockdown/knockout conditions

  • Compare wild-type and mutant substrate proteins for ubiquitination-site mapping

  • Include ubiquitin mutants to identify specific chain types (e.g., K48, K63, K29 linkages)

HECTD1 has been reported to assemble both proteasomal (K48-linked) and non-proteasomal (K63-linked) ubiquitin signals, so experimental approaches should be capable of distinguishing these outcomes .

What are common challenges in detecting HECTD1 by western blotting?

HECTD1 detection presents several challenges due to its large size (~290 kDa) and potential for degradation:

• Size-related issues:

  • Use lower percentage gels (6-8%) for better resolution of high molecular weight proteins

  • Extend transfer time (overnight at lower voltage) to ensure complete transfer of large proteins

  • Consider using gradient gels for better separation

• Protein degradation concerns:

  • Include protease inhibitors in all buffers

  • Maintain samples at 4°C during preparation

  • When studying HECTD1 cleavage during apoptosis, be aware that HECTD1 is cleaved upon induction of both intrinsic and extrinsic apoptotic pathways

• Signal optimization:

  • Recommended antibody dilutions for western blotting range from 1:500 to 1:3000

  • Normalize loading with appropriate controls (GAPDH is commonly used)

  • Extended blocking (2 hours or overnight) may reduce background

  • If detecting endogenous HECTD1, consider longer exposure times due to potentially low expression levels

• Verification strategies:

  • When evaluating antibody specificity, compare wild-type samples with HECTD1 knockout/knockdown samples

  • If possible, include multiple HECTD1 antibodies targeting different epitopes

How can I distinguish between different functional domains of HECTD1 in experimental systems?

HECTD1 contains multiple functional domains that contribute to its diverse cellular roles:

• Domain-specific analysis approaches:

  • Use domain-swapping mutagenesis to study specific functional regions

  • For example, fusing ubiquitin to the N-terminus of C-terminal HECTD1 fragments can be used to test fragment stability

  • Generate truncated constructs that separate substrate binding domains from the catalytic HECT domain

• Functional domain analysis using mutant models:

  • The HECTD1 XC mouse line expresses a truncated HECTD1 protein retaining substrate binding domains but with a disrupted C-terminal ubiquitin ligase domain

  • The HECTD1 opm mouse line harbors an ENU-induced nonsense mutation that truncates the 2610-amino-acid HECTD1 protein after amino acid 144

  • These models allow separation of binding function from catalytic activity

• Detecting domain-specific interactions:

  • For subcellular localization analysis, use prediction software such as RSLpred and PSORT II

  • When studying nuclear-cytoplasmic shuttling, treatments with nuclear export inhibitors like Leptomycin B (LMB, 50 nM) or ivermectin (IVE, 1 μM) can help determine functional domain requirements

  • Perform domain mapping through co-immunoprecipitation with truncated constructs

Researchers should note that different functional domains may contribute to distinct cellular processes, with the HECT domain being critical for ubiquitin ligase activity but not necessarily for protein-protein interactions .

How can I design experiments to study HECTD1's role in cell proliferation and mitosis?

Based on established research findings, HECTD1 contributes to cell proliferation through regulation of mitosis:

• Cellular model selection:

  • HEK293T and HeLa cells are validated models for HECTD1 studies in cell proliferation

  • Both transient knockdown and genetic knockout approaches have been successful

• Experimental design framework:

  • Cell proliferation assays:

    • Compare wild-type with HECTD1-depleted cells using cell counting, MTT/XTT assays, or real-time growth monitoring

    • Include rescue experiments with wild-type HECTD1 and ligase-deficient HECTD1 C2579G to determine if proliferation effects are mediated through ubiquitin ligase activity

  • Mitotic phase analysis:

    • Quantify cells with aligned chromosomes (Prometa/Metaphase)

    • Use phospho-Histone H3 (Ser28) as a molecular marker of mitosis

    • Perform time-lapse microscopy of nuclear envelope breakdown (NEBD) to anaphase onset to measure mitotic timing

  • Spindle assembly checkpoint evaluation:

    • Analyze BUB3, BUBR1, and MAD2 protein levels

    • Consider immunoprecipitation experiments to detect interaction between HECTD1 and components of the Mitotic Checkpoint Complex like BUB3

• Critical controls:

  • Include both wild-type and ligase-deficient HECTD1 constructs in rescue experiments

  • Compare multiple HECTD1 knockout/knockdown clones to rule out off-target effects

  • Verify mitotic timing alterations with multiple methodological approaches

HECTD1 depletion has been shown to increase the proportion of cells with aligned chromosomes and extend the time from NEBD to anaphase onset, suggesting a role in mitotic progression regulation .

What methodologies are recommended for studying HECTD1 in hematopoietic stem cell research?

HECTD1 plays a critical role in hematopoietic stem cell (HSC) function and regeneration:

• In vivo experimental approaches:

  • Competitive bone marrow transplantation assay:

    • Inject bone marrow cells from HECTD1-deficient donor mice (CD45.2) with equal numbers of competitor cells (CD45.1) into lethally irradiated recipients

    • Analyze donor chimerism in peripheral blood by flow cytometry every 4 weeks

    • Determine HSC frequencies using extreme limiting dilution analysis (ELDA)

  • Purified HSC transplantation:

    • Isolate HSCs (LSK CD150+CD48−) by flow cytometric sorting

    • Transplant defined numbers of cells into lethally irradiated recipients with competitors

    • Compare reconstitution ability between wild-type and HECTD1-deficient HSCs

    • This approach allows distinction between intrinsic HSC defects and microenvironment effects

• Ex vivo culture systems:

  • Culture purified HSCs in media containing cytokines (SCF, TPO, FLT3L, IL6)

  • Track cell growth, differentiation, and maintenance of stem cell identity

  • Perform transplantation of cultured cells to test functional preservation

  • Compare freshly isolated vs. cultured HSCs for reconstitution potential

• Human cell line models:

  • TF-1 cells can be used as a reliable human model system

  • Evaluate growth in the presence of TPO or GM-CSF upon HECTD1 knockdown

  • Assess signaling pathways, particularly RPS6 phosphorylation

Research has demonstrated that HECTD1 deficiency decreases both HSC frequency and function in vivo, with particularly pronounced effects on HSC maintenance during ex vivo culture .

How can I investigate HECTD1's interaction with specific substrates like RARA or SNAIL?

HECTD1 regulates multiple substrates through ubiquitination, including RARA (retinoic acid receptor alpha) and SNAIL:

• Substrate-specific detection methods:

  • RARA interaction studies:

    • Co-immunoprecipitate HECTD1 and RARA under various conditions

    • Analyze ubiquitination patterns using FK1 (polyubiquitin-specific) and FK2 (mono- and polyubiquitin) antibodies

    • Compare wild-type and ligase-deficient HECTD1 effects on RARA stability and ubiquitination

    • Assess the impact on retinoic acid signaling using reporter assays

  • SNAIL regulation analysis:

    • Perform CHX chase assays to measure SNAIL protein stability

    • Treat cells with leptomycin B (LMB) or ivermectin (IVE) to analyze nuclear-cytoplasmic shuttling

    • Use immunofluorescence to quantify nuclear vs. cytoplasmic SNAIL distribution in the presence or absence of HECTD1

• Functional outcome assessment:

  • For RARA-mediated pathways:

    • Evaluate embryonic patterning defects in HECTD1 mutant models

    • Analyze expression of retinoic acid target genes

    • Assess phenotypes associated with retinoic acid signaling such as aortic arch development

  • For SNAIL-mediated processes:

    • Measure cell migration using wound healing or transwell migration assays

    • Assess epithelial-mesenchymal transition (EMT) markers

    • Evaluate expression of SNAIL target genes

• Critical controls for substrate studies:

  • Include ubiquitin ligase-deficient HECTD1 (C2579G) to distinguish between binding and catalytic effects

  • Use proteasome inhibitors to stabilize ubiquitinated species

  • Consider miRNA regulation as an alternative mechanism of substrate control

Research has shown that HECTD1 interacts with RARA and influences its ubiquitination state, with HECTD1 deficiency leading to reduced retinoic acid signaling in embryos. Similarly, HECTD1 regulates SNAIL stability through ubiquitination, with HECTD1 knockdown increasing SNAIL expression levels .

What are the methodological considerations for studying HECTD1 in apoptotic pathways?

Recent research has identified HECTD1 as both a regulator and a substrate in apoptotic pathways:

• Experimental design for HECTD1 cleavage studies:

  • Induce apoptosis through intrinsic or extrinsic pathways

  • Monitor HECTD1 cleavage using antibodies that can detect both full-length and cleaved fragments

  • Verify cleavage site identification through mutagenesis

  • Use domain-swapping mutagenesis (e.g., fusing ubiquitin to the N-terminus of C-terminal HECTD1 fragments) to study fragment stability

• Caspase-mediated regulation:

  • Identify specific caspases involved in HECTD1 cleavage using caspase inhibitors or knockout/knockdown approaches

  • Analyze the functional implications of HECTD1 cleavage on its ubiquitin ligase activity

  • Determine whether cleaved HECTD1 fragments retain specific functions or act as dominant negatives

• Technical considerations:

  • Ensure sufficient separation of high molecular weight proteins when analyzing HECTD1 cleavage

  • Include appropriate controls for apoptosis induction

  • Consider temporal dynamics of HECTD1 cleavage in relation to other apoptotic events

  • Analyze subcellular localization changes of HECTD1 fragments after cleavage

Research has demonstrated that HECTD1 is cleaved during apoptosis and may serve as both a regulator and substrate of caspase-3, suggesting a complex role in cell death pathways .

How can mass spectrometry-based approaches enhance HECTD1 research?

Mass spectrometry (MS) provides powerful tools for HECTD1 research:

• Identification of interaction partners:

  • Perform affinity purification of HECTD1 complexes followed by MS

  • Express HA-tagged HECTD1 in appropriate cell systems

  • Use HA peptide affinity elution to minimize non-specific binding

  • Analyze results using the Contaminant Repository for Affinity Purification (CRAPome) to filter out common contaminants

  • Validate MS-identified interactions through orthogonal methods

• Ubiquitination site mapping:

  • Identify specific lysine residues on substrates that are ubiquitinated by HECTD1

  • Distinguish between different ubiquitin chain types (K48, K63, K29)

  • Quantify changes in ubiquitination patterns upon HECTD1 manipulation

• Technical considerations:

  • Perform experiments in triplicate to ensure reproducibility

  • Include appropriate negative controls (vector alone) for comparison

  • Verify efficiency of purification through sequential elution steps

  • Consider using SILAC or TMT labeling for quantitative analysis of HECTD1-dependent changes

This approach has successfully identified HECTD1 interaction partners in hematopoietic cells, revealing connections to ribosome assembly factors like ZNF622 .