OTUD6B Antibody

What is OTUD6B and what are its primary cellular functions?

OTUD6B (OTU domain-containing protein 6B, also known as DUBA5 or CGI-77) is a deubiquitinating enzyme belonging to the OTU domain superfamily of cysteine proteases. It specifically targets ubiquitin chains on substrate proteins for removal, which regulates protein stability and activity across various cellular processes . OTUD6B contains a C-terminal catalytic domain and three coiled-coil domains at the N-terminus that may mediate protein interactions .

The primary cellular functions of OTUD6B include:

• Regulation of protein synthesis downstream of mTORC1, potentially by associating with protein synthesis initiation complexes and modifying their ubiquitination to repress translation

• Regulation of cell growth and proliferation by repressing DNA synthesis and modifying different cellular targets

• Involvement in proteasome assembly and function

• Positive regulation of IRF3-mediated antiviral immune responses through stabilization of IRF3 by hydrolyzing Lys33-linked polyubiquitin chains

• Regulation of KIFC1-dependent centrosome clustering, which is critical for survival of cancer cells with centrosome amplification

Interestingly, different isoforms of OTUD6B can have opposite effects. While isoform 1 may repress translation, isoform 2 has been reported to stimulate protein synthesis, influence CCND1/cyclin D1 expression, and regulate MYC/c-Myc protein stability .

What types of OTUD6B antibodies are available for research, and what epitopes do they target?

Currently available OTUD6B antibodies include rabbit polyclonal antibodies suitable for various research applications. Based on the search results, these antibodies target different regions of the OTUD6B protein:

• Antibodies targeting the full-length human OTUD6B protein (recombinant full-length protein immunogen)

• Antibodies targeting fragments within the human OTUD6B protein (e.g., from amino acid 50 to the C-terminus)

These antibodies have been validated for various applications including Western blot (WB), immunohistochemistry on paraffin-embedded sections (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) . They have been tested primarily against human samples due to the specificity of the antibodies for human OTUD6B .

How is OTUD6B protein structure related to its function, and what should researchers consider when selecting antibodies?

OTUD6B's structure includes an OTU domain responsible for its catalytic deubiquitinating activity and multiple coiled-coil domains that mediate protein-protein interactions . The N-terminal domain is particularly important as it has been shown to interact with substrates such as KIFC1, independent of OTUD6B's catalytic activity .

When selecting antibodies for OTUD6B research, researchers should consider:

• The specific isoform of interest: OTUD6B has multiple isoforms with potentially opposing functions

• The domain of interest: Antibodies targeting different epitopes may be more suitable depending on whether the research focuses on the catalytic activity or protein interactions

• Post-translational modifications: Some modifications might mask epitopes and affect antibody binding

• Intended application: Ensure the antibody has been validated for the specific technique (WB, IHC-P, ICC/IF) to be used

• Species cross-reactivity: Most available antibodies are validated for human samples, so cross-reactivity with other species should be verified if studying non-human models

What are the optimal conditions for Western blot detection of OTUD6B?

For optimal Western blot detection of OTUD6B using commercially available antibodies, the following conditions are recommended based on the search results:

Sample preparation and loading:

• Cell lysates from human cell lines such as PC-3, HEK-293T, or MDA-MB-231 have been successfully used

• The predicted molecular weight of OTUD6B is approximately 33 kDa, but the observed band size is typically around 34 kDa

Antibody concentrations and detection:

• Primary antibody (e.g., ab235326) can be used at a concentration of approximately 4 μg/mL

• Secondary antibody (e.g., Goat polyclonal to Rabbit IgG) can be used at a 1/10000 dilution

• For detection systems, standard chemiluminescence or fluorescence-based methods are appropriate

Controls and validation:

• Positive controls should include cell lines known to express OTUD6B (such as HEK-293T)

• Negative controls should include knockdown/knockout samples or isotype controls

• Validation of specificity can be performed using siRNA-mediated knockdown of OTUD6B, which has been demonstrated to effectively reduce OTUD6B protein levels in previous studies

How can OTUD6B be detected in tissue samples using immunohistochemistry?

For immunohistochemical detection of OTUD6B in tissue samples:

Sample preparation:

• Paraffin-embedded tissue sections are suitable for OTUD6B detection

• Standard deparaffinization, rehydration, and antigen retrieval steps should be performed

Staining protocol:

• Based on published methods, an antibody dilution of 1/100 has been successfully used for OTUD6B detection in paraffin-embedded human tissues

• Both human ovarian cancer tissue and small intestine tissue have been successfully stained for OTUD6B

Optimization considerations:

• Antigen retrieval methods may need optimization depending on tissue fixation

• Blocking steps should be included to reduce background staining

• Secondary antibody selection should be compatible with the detection system

• Both chromogenic and fluorescent detection systems can be employed depending on the research question

Analysis and interpretation:

• OTUD6B expression patterns should be evaluated in the context of relevant subcellular localization (which may include centrosomes and mitotic spindles during cell division)

• Comparison with normal tissues is important, especially when studying cancer samples, as OTUD6B is commonly overexpressed in certain cancers like breast cancer

What approaches can be used to study OTUD6B localization within cells?

To study OTUD6B localization within cells, several complementary approaches can be employed:

Immunocytochemistry/Immunofluorescence (ICC/IF):

• Fixed cells can be stained with anti-OTUD6B antibodies that have been validated for ICC/IF applications

• Co-staining with markers for specific cellular structures (such as centrosomes, mitotic spindle, or proteasomes) can reveal functional associations

• Research has shown that OTUD6B can localize to centrosomes and the mitotic spindle in certain contexts

Subcellular fractionation:

• Biochemical separation of cellular compartments followed by Western blotting can quantitatively assess OTUD6B distribution

• Nuclear, cytoplasmic, and organelle-enriched fractions should be analyzed

• Controls for fraction purity should be included (e.g., lamin for nuclear fraction, tubulin for cytoskeletal fraction)

Live-cell imaging:

• Expression of GFP-tagged OTUD6B has been successfully used to study its localization and interactions

• Time-lapse imaging can reveal dynamic localization changes during cell cycle progression or in response to stimuli

• Care should be taken to ensure that tagging does not interfere with protein function

Electron microscopy:

• For ultra-high resolution studies of OTUD6B localization, immunogold labeling combined with electron microscopy can be employed

• This approach is particularly useful for precise localization within complex structures like centrosomes

How does OTUD6B regulate antiviral immune responses, and what experimental models are suitable for studying this function?

OTUD6B plays a significant role in regulating antiviral immune responses, particularly through the type I interferon (IFN) pathway. Research has demonstrated that human OTUD6B positively regulates IRF3-mediated antiviral immune responses through specific mechanisms:

Mechanisms of action:

• OTUD6B interacts directly with IRF3 (Interferon Regulatory Factor 3), a key transcription factor for type I IFN production

• It specifically hydrolyzes K33-linked polyubiquitin chains at Lys315 of IRF3, which stabilizes IRF3 protein levels

• By preventing IRF3 degradation, OTUD6B enhances type I IFN production and downstream antiviral responses

• This is notably different from zebrafish OTUD6B, which negatively regulates the antiviral response by suppressing K63-linked ubiquitination of IRF3 and IRF7

Experimental models for studying OTUD6B's role in antiviral immunity:

• Cell culture models:

  • Human cell lines such as 293T, HT1080, HeLa, and Hep2 have been successfully used

  • Knockdown approaches using siRNA targeting OTUD6B or overexpression systems with HA-tagged OTUD6B

  • Viral infection models including VSV, H1N1, SeV, RSV, and HSV-1 have demonstrated broad-spectrum antiviral effects of OTUD6B

• In vivo models:

  • Transgenic mice overexpressing human OTUD6B show enhanced resistance to RNA virus infection with reduced viral load and morbidity

  • Wild-type mice can be compared with OTUD6B-overexpressing mice to assess antiviral responses

• Functional assays:

  • Luciferase reporter assays using IFN-β promoter or IFN-stimulatory response element constructs

  • qPCR analysis of IFN-β and interferon-stimulated gene (ISG) mRNA levels

  • ELISA detection of secreted IFN-β in cell culture medium

  • Viral replication assays using reporter viruses or quantitative PCR for viral genomes

What are the methodological approaches to study OTUD6B's deubiquitinating activity on IRF3?

To investigate OTUD6B's deubiquitinating activity on IRF3 and other potential substrates, several specialized methodological approaches can be employed:

Co-immunoprecipitation (Co-IP) and protein interaction studies:

• Exogenous expression of tagged OTUD6B and IRF3 followed by immunoprecipitation can demonstrate direct interaction

• Pull-down of endogenous proteins can confirm physiologically relevant interactions

• Domain mapping using truncation mutants can identify specific interaction regions

Ubiquitination assays:

• In vitro deubiquitination assays using purified components (OTUD6B, ubiquitinated IRF3)

• Cellular ubiquitination assays comparing IRF3 ubiquitination in the presence or absence of OTUD6B

• Ubiquitin chain-specific antibodies can distinguish between different ubiquitin linkage types (K11, K33, K48, K63)

• Site-directed mutagenesis of IRF3 (e.g., K315R mutation) can confirm specific ubiquitination sites

Enzymatic activity analysis:

• Catalytically dead mutants (e.g., OTUD6B-C158S) can be used as negative controls

• Ubiquitin activity probes (e.g., HA-Ub-PA) can confirm OTUD6B catalytic activity

• Mass spectrometry analysis of ubiquitin chain types before and after OTUD6B treatment

Functional readouts:

• IRF3 protein stability assays (protein half-life determination using cycloheximide chase)

• IRF3 nuclear translocation assays following viral stimulation

• IRF3-dependent gene expression analysis (qPCR, reporter assays)

• Type I IFN production and antiviral activity measurements

How can researchers distinguish between OTUD6B's effects on different signaling pathways in immune cells?

Distinguishing between OTUD6B's effects on different signaling pathways in immune cells requires careful experimental design and specific pathway inhibition or activation approaches:

Pathway-specific stimulation:

• Use specific pathway activators/inducers:

  • RIG-I pathway: 5'ppp-dsRNA, SeV infection

  • cGAS-STING pathway: dsDNA, cGAMP

  • TLR pathways: Specific TLR ligands (LPS, poly(I:C), CpG DNA, etc.)

• Monitor pathway-specific outputs following OTUD6B manipulation

Genetic approaches:

• Generate knockout/knockdown of key components in each pathway alongside OTUD6B manipulation

• Create cell lines with pathway-specific reporter constructs to monitor specific signaling outputs

• Use CRISPR-Cas9 to generate OTUD6B-deficient immune cell lines for pathway analysis

Biochemical approaches:

• Analyze phosphorylation status of pathway-specific components (e.g., TBK1, IRF3, NF-κB)

• Perform proximity ligation assays to detect specific OTUD6B interactions with pathway components

• Use specific pathway inhibitors alongside OTUD6B modulation

Transcriptomic and proteomic analysis:

• RNA-seq or microarray analysis to identify pathway-specific gene expression signatures

• Phosphoproteomics to identify pathway-specific phosphorylation events

• Systematic analysis of ubiquitination changes in different pathway components

Functional outcomes:

• Measure cytokine profiles characteristic of different pathways

• Assess cellular responses to different pathogens that predominantly activate specific pathways

• Evaluate immune cell differentiation, activation, or effector functions

What is the role of OTUD6B in centrosome clustering, and how can this be experimentally investigated?

OTUD6B plays a critical role in centrosome clustering in cancer cells with supernumerary centrosomes, particularly in triple-negative breast cancer (TNBC):

Mechanism of action:

• OTUD6B functions as a deubiquitinase that stabilizes KIFC1 (also known as HSET), a kinesin motor protein essential for centrosome clustering

• OTUD6B can localize to centrosomes and the mitotic spindle during cell division

• It directly interacts with KIFC1 through its N-terminal domain (independent of catalytic activity)

• OTUD6B prevents excessive polyubiquitination and premature degradation of KIFC1 during mitosis

• This activity allows cancer cells with centrosome amplification to form pseudo-bipolar spindles rather than lethal multipolar spindles

Experimental approaches to investigate OTUD6B's role in centrosome clustering:

• Imaging-based approaches:

  • Immunofluorescence microscopy to visualize centrosome numbers and clustering using centrosome markers (γ-tubulin, pericentrin) in cells with modulated OTUD6B levels

  • Quantification of multipolar spindle frequency in mitotic cells

  • Live-cell imaging to track centrosome dynamics during mitosis in real-time

  • Super-resolution microscopy for detailed analysis of centrosome structure

• Molecular and biochemical approaches:

  • siRNA or CRISPR-Cas9-mediated depletion of OTUD6B to assess effects on KIFC1 levels and centrosome clustering

  • Co-immunoprecipitation to confirm OTUD6B-KIFC1 interaction

  • Ubiquitination assays to measure KIFC1 ubiquitination levels with or without OTUD6B

  • Expression of catalytically inactive OTUD6B (C158S mutant) to determine enzyme activity requirements

  • Rescue experiments with KIFC1 overexpression in OTUD6B-depleted cells

• Functional assays:

  • Cell proliferation and survival assays in cancer cells with centrosome amplification versus normal cells

  • Colony formation assays to assess long-term effects of OTUD6B depletion

  • Cell cycle analysis to identify mitotic arrest or delays

  • Apoptosis assays to quantify cell death following disruption of centrosome clustering

How does OTUD6B expression correlate with cancer progression, and what techniques are most reliable for analyzing this correlation?

OTUD6B expression has been found to correlate with cancer progression, particularly in breast cancer:

Expression patterns and correlations:

• OTUD6B is commonly overexpressed in breast cancer

• Its expression correlates with KIFC1 protein expression in breast cancer tissues

• Higher OTUD6B expression is associated with worse patient survival in breast cancer

• Triple-negative breast cancer (TNBC) cells with centrosome amplification depend on OTUD6B for proliferation and survival

Techniques for analyzing OTUD6B-cancer correlations:

• Expression analysis in clinical samples:

  • Immunohistochemistry (IHC) on tissue microarrays to quantify OTUD6B expression across cancer stages and subtypes

  • Western blot analysis of patient-derived samples

  • qRT-PCR for mRNA expression analysis in patient cohorts

  • RNA-seq data analysis from public databases (TCGA, METABRIC)

• Survival and clinical correlation studies:

  • Kaplan-Meier survival analysis stratifying patients by OTUD6B expression levels

  • Multivariate analysis to adjust for confounding clinical variables

  • Correlation with established clinical parameters (stage, grade, receptor status)

  • Meta-analysis across multiple patient cohorts

• Functional validation:

  • CRISPR-Cas9 genetic editing to create OTUD6B knockout cancer cell lines

  • Xenograft models comparing tumor growth with or without OTUD6B expression

  • Patient-derived organoids with OTUD6B modulation

  • Analysis of therapy response in relation to OTUD6B expression

• Mechanistic studies:

  • Correlation of OTUD6B with centrosome amplification markers

  • Co-expression analysis with KIFC1 and other mitotic regulators

  • Analysis of downstream pathways affected by OTUD6B overexpression

  • Cell type-specific effects (cancer cells versus normal epithelial cells)

What experimental models are most appropriate for studying OTUD6B as a potential therapeutic target in cancer?

Given OTUD6B's role in supporting cancer cell survival, particularly in cells with centrosome amplification, several experimental models can be employed to evaluate its potential as a therapeutic target:

Cell line models:

• Triple-negative breast cancer (TNBC) cell lines with naturally occurring centrosome amplification (e.g., BT549)

• Normal breast epithelial cell lines as controls to assess cancer-specific effects

• Isogenic cell line pairs differing only in OTUD6B expression

• Cell lines engineered to express OTUD6B variants (wild-type, catalytically inactive, domain deletions)

• Doxycycline-inducible OTUD6B knockdown/overexpression systems for temporal control

Animal models:

• Xenograft models using OTUD6B-manipulated cancer cell lines

• Genetically engineered mouse models (GEMMs) with conditional OTUD6B knockout in specific tissues

• Patient-derived xenograft (PDX) models with varying levels of centrosome amplification

• Syngeneic mouse models for studying OTUD6B in the context of an intact immune system

Ex vivo models:

• Patient-derived organoids

• Tissue slice cultures

• 3D spheroid models to better recapitulate tumor architecture

Drug development approaches:

• High-throughput screening for small molecule inhibitors of OTUD6B catalytic activity

• Structure-based drug design targeting the OTUD6B catalytic domain

• Peptide-based inhibitors disrupting OTUD6B-KIFC1 interaction

• Testing OTUD6B inhibition in combination with conventional chemotherapies or targeted agents

• Synthetic lethality screens to identify genes that, when inhibited alongside OTUD6B, lead to enhanced cancer cell death

Biomarker development:

• Correlation of centrosome amplification status with OTUD6B dependency

• Identification of molecular signatures predicting response to OTUD6B inhibition

• Development of companion diagnostics for patient stratification

What are common technical challenges when working with OTUD6B antibodies, and how can they be addressed?

Researchers working with OTUD6B antibodies may encounter several technical challenges:

Specificity issues:

• Cross-reactivity with other OTU family proteins due to domain conservation

• Detection of multiple isoforms with potentially different functions

• Non-specific binding in certain tissues or cell types

Solutions:

• Validate antibody specificity using OTUD6B knockdown/knockout controls

• Compare results from multiple antibodies targeting different epitopes

• Include appropriate blocking steps to reduce non-specific binding

• Perform preabsorption controls with recombinant OTUD6B protein

Signal strength and detection:

• Weak signal in certain applications or tissues

• High background staining

• Variable expression levels across different cell types

Solutions:

• Optimize antibody concentration through titration experiments

• Test different antigen retrieval methods for IHC applications

• Use signal amplification methods (e.g., tyramide signal amplification)

• Explore alternative detection systems (chemiluminescence, fluorescence)

• Concentrate protein samples when working with low-expressing cells

Reproducibility challenges:

• Batch-to-batch variation in antibody performance

• Inconsistent results across different experimental conditions

• Differences between fresh and frozen samples

Solutions:

• Maintain consistent experimental conditions (fixation times, buffer compositions)

• Document lot numbers and maintain reference samples for comparison

• Use standardized positive controls across experiments

• Consider monoclonal antibodies for greater consistency once validated

How can researchers effectively design experiments to study OTUD6B's catalytic activity and substrate specificity?

Designing experiments to study OTUD6B's catalytic activity and substrate specificity requires careful consideration of enzyme properties, reaction conditions, and detection methods:

Enzyme activity assays:

• In vitro deubiquitination assays using recombinant OTUD6B and synthetic ubiquitin chains

• Activity-based probes (like HA-Ub-PA) to assess catalytic site functionality

• Fluorogenic substrate assays for quantitative activity measurements

• Include catalytically inactive mutants (e.g., C158S) as negative controls

Substrate identification and validation:

• Proteomic approaches:

  • Stable isotope labeling by amino acids in cell culture (SILAC) combined with mass spectrometry

  • Ubiquitin remnant profiling comparing wild-type and OTUD6B-depleted cells

  • Proximity-based labeling (BioID, APEX) to identify proteins in OTUD6B vicinity

• Candidate approach:

  • Co-immunoprecipitation followed by ubiquitination analysis

  • Targeted analysis of potential substrates (e.g., IRF3, KIFC1)

  • In vitro deubiquitination of purified ubiquitinated candidate substrates

Ubiquitin chain specificity:

• Analysis with linkage-specific ubiquitin antibodies (K11, K33, K48, K63)

• Mass spectrometry to identify precise ubiquitin linkage types

• In vitro assays with defined ubiquitin chain types

• Ubiquitin mutant panels to assess linkage preferences

Domain and structural requirements:

• Structure-function analysis using truncation mutants

• Point mutations in catalytic and substrate-binding domains

• Chimeric constructs exchanging domains between OTUD6B and related DUBs

• Computational modeling and molecular dynamics simulations

Cellular contexts and regulation:

• Cell cycle-dependent activity assays (particularly during mitosis)

• Analysis under different stress conditions (viral infection, proteotoxic stress)

• Phosphorylation status effects on OTUD6B activity

• Subcellular localization effects on substrate accessibility

What is the best approach to integrate OTUD6B research findings across different biological contexts (immunity vs. cancer)?

Integrating OTUD6B research findings across different biological contexts requires a systematic approach that considers common mechanisms while acknowledging context-specific functions:

Comparative mechanistic analysis:

• Systematically compare OTUD6B's molecular interactions across contexts

  • In immunity: IRF3 stabilization through K33-linked deubiquitination

  • In cancer: KIFC1 stabilization supporting centrosome clustering

• Identify common principles in substrate recognition and regulation

• Determine if the same catalytic mechanisms operate in different contexts

Multi-omics integration:

• Perform transcriptomic, proteomic, and ubiquitinome analyses in both immune and cancer contexts

• Use computational approaches to identify common and distinct pathways

• Network analysis to map OTUD6B's position in different cellular processes

• Temporal profiling to understand dynamic regulation across contexts

Unified experimental systems:

• Develop cell systems where both functions can be studied simultaneously

• Use inducible expression/depletion systems to examine context-dependent effects

• Apply consistent methodologies across research areas for direct comparability

• Develop reporter systems that can monitor multiple OTUD6B functions

Translational integration:

• Examine clinical datasets for correlations between OTUD6B expression and both immune and cancer phenotypes

• Consider how OTUD6B targeting might simultaneously affect immune response and cancer cell survival

• Develop experimental models to test dual-context effects (e.g., immune-competent cancer models)

Collaborative research framework:

• Establish collaborations between immunology and cancer research groups

• Standardize reagents and methodologies across disciplines

• Create shared databases of OTUD6B interactions, modifications, and activities

• Develop integrated research questions that span biological contexts

This integrated approach will help resolve apparent contradictions in OTUD6B functions and provide a more complete picture of its biological roles and therapeutic potential.

What are the most promising future research directions for OTUD6B antibody applications?

The emerging understanding of OTUD6B's diverse functions in both immunity and cancer biology points to several promising research directions for OTUD6B antibody applications:

Advanced imaging applications:

• Super-resolution microscopy to precisely localize OTUD6B at centrosomes and mitotic spindles

• Live-cell imaging using cell-permeable antibody fragments or nanobodies

• Correlative light and electron microscopy (CLEM) for ultrastructural analysis

• Expansion microscopy for enhanced visualization of OTUD6B in complex structures

Therapeutic development:

• Antibody-based disruption of specific OTUD6B interactions (e.g., with KIFC1 in cancer cells)

• Intrabody approaches to target OTUD6B in specific cellular compartments

• Antibody-drug conjugates targeting cancer cells with high OTUD6B expression

• Development of conformation-specific antibodies that distinguish active vs. inactive OTUD6B

Diagnostic applications:

• Multiplex immunohistochemistry panels combining OTUD6B with other cancer markers

• Liquid biopsy approaches to detect OTUD6B in circulating tumor cells

• Prognostic and predictive biomarker development for cancer patient stratification

• Immune monitoring during viral infections or immunotherapy

Technical innovations:

• Development of antibodies specific to post-translationally modified OTUD6B

• Isoform-specific antibodies to distinguish between functionally distinct variants

• Nanobodies or single-domain antibodies for improved penetration and reduced immunogenicity

• Proximity labeling antibodies to identify context-specific OTUD6B interactors