USP18 Antibody

What is USP18 and what are its primary functions in cellular pathways?

USP18 is a ubiquitin-specific peptidase that serves multiple crucial functions in cellular processes. It has two primary functional domains: one responsible for isopeptidase activity and another that inhibits type I interferon signaling by blocking the type I interferon receptor 2 (IFNAR2) subunit . As an isopeptidase, USP18 removes ISG15 (interferon-stimulated gene 15) from conjugated proteins, thus regulating the ISGylation process. Its inhibitory effect on interferon signaling has significant implications for viral infections, immune cell development, and autoimmune diseases .

USP18 plays a role in the development of CD11b+ dendritic cells and Th17 cells, contributing to immune system regulation . Additionally, it has been implicated in tumorigenesis through various mechanisms including cell proliferation regulation and influencing the tumor microenvironment . The multifunctional nature of USP18 makes it a valuable target for research across immunology, virology, and oncology fields.

What are the most common applications for USP18 antibodies in research?

USP18 antibodies are employed in numerous research applications, with the most commonly used techniques being Western Blotting (WB), Enzyme-Linked Immunosorbent Assay (ELISA), Immunofluorescence (IF), Immunohistochemistry (IHC), and Immunoprecipitation (IP) . These applications allow researchers to detect, quantify, localize, and study the interactions of USP18 in various experimental contexts.

Western Blotting is frequently used to detect USP18 protein expression levels in cell or tissue lysates, while ELISA provides quantitative measurement of USP18 in solution. Immunofluorescence and immunohistochemistry enable visualization of USP18 distribution within cells or tissues, respectively. Immunoprecipitation is valuable for studying USP18 protein interactions or for isolating USP18 complexes from cell lysates . The choice of application depends on the specific research question, with many studies employing multiple techniques to strengthen their findings.

How does USP18 impact interferon signaling pathways?

USP18 serves as a potent negative regulator of type I interferon (IFN-I) signaling by specifically blocking the IFNAR2 subunit of the type I interferon receptor . In the absence of USP18, phosphorylation of STAT1 and STAT2 is prolonged, leading to enhanced expression of hundreds of antiviral genes, chemokines, cytokines, and antigen-presenting genes . This regulatory function is independent of USP18's isopeptidase activity, as demonstrated by studies using the C61A mutant that lacks enzymatic activity but still inhibits IFN-I signaling .

What criteria should be considered when selecting a USP18 antibody for specific applications?

When selecting a USP18 antibody for research, several critical factors should be evaluated to ensure experimental success:

Reactivity and Species Specificity: Verify that the antibody recognizes USP18 from your species of interest (human, mouse, rat, etc.) . Cross-reactivity between species can be beneficial for comparative studies but may cause interpretation challenges if unwanted.

Application Compatibility: Confirm the antibody has been validated for your intended application (WB, ELISA, IF, IHC, or IP) . An antibody that works well for Western blotting may not necessarily perform optimally for immunohistochemistry.

Clonality: Monoclonal antibodies (like the D4E7 rabbit mAb) offer high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes and may provide stronger signals but potentially more background .

Conjugation: Determine whether you need an unconjugated antibody or one conjugated to enzymes (HRP), fluorophores, or agarose for immunoprecipitation .

Validation Evidence: Review available data showing the antibody's performance, including published figures, citations, and validation data from suppliers . Antibodies with extensive publication history generally offer more reliable performance.

Epitope Location: Consider which domain of USP18 you wish to target—antibodies recognizing different epitopes may yield different results, especially if studying specific functional domains of USP18 .

How can I optimize Western blot protocols for detecting USP18 in different cell types?

Optimizing Western blot protocols for USP18 detection requires careful consideration of several parameters:

Lysate Preparation: Different cell types may require modified lysis buffers. For USP18 detection, use buffers containing protease inhibitors to prevent degradation, and consider adding deubiquitinase inhibitors to preserve USP18's native state .

Protein Loading: USP18 expression varies substantially between cell types and can be significantly upregulated by interferon stimulation. For baseline detection in unstimulated cells, higher protein amounts (30-50μg) may be necessary, while interferon-stimulated samples may require less (10-20μg) to avoid signal saturation .

Antibody Concentration: Titrate primary antibody concentrations, typically starting with the manufacturer's recommendation (often 1:1000) and adjusting as needed. The D4E7 rabbit monoclonal antibody has shown good results in detecting USP18 in human cell line lysates .

Detection Method: Enhanced chemiluminescence (ECL) systems offer good sensitivity for USP18 detection. For low expression scenarios, consider using more sensitive detection methods like ECL-Plus or fluorescence-based detection systems.

Controls: Always include positive controls (interferon-stimulated cells known to express USP18) and negative controls (USP18 knockout cells if available). For comparing expression levels between conditions, verify equal loading using housekeeping proteins like GAPDH or β-actin.

Membrane Blocking: Optimize blocking conditions (typically 5% non-fat milk or BSA) to reduce background while maintaining specific signal. Some USP18 antibodies may perform better with one blocking agent over another.

What experimental controls are essential when studying USP18 expression or function?

Rigorous experimental design for USP18 studies necessitates several important controls:

Positive Expression Controls: Include samples with known USP18 expression, such as cells treated with type I interferons, which strongly induce USP18 expression . This confirms antibody functionality and system responsiveness.

Negative Controls: Where possible, use USP18 knockout cells or tissues as negative controls to confirm antibody specificity . If knockouts aren't available, siRNA or shRNA knockdown samples provide valuable alternatives.

Isotype Controls: For immunostaining applications, include appropriate isotype controls matched to your primary antibody to distinguish specific staining from non-specific binding or Fc receptor interactions.

Functional Domain Controls: When studying specific USP18 functions, consider using the C61A mutant that lacks isopeptidase activity but retains interferon regulation capability . This allows differentiation between USP18's dual functions.

Loading Controls: For expression studies, always include appropriate loading controls (β-actin, GAPDH, etc.) to normalize USP18 levels and enable accurate comparisons between conditions.

Time Course Controls: Since USP18 expression is highly dynamic and regulated by interferons, including time course samples after stimulation helps establish proper experimental timing and captures expression peaks.

Treatment Validation: When using interferon stimulation, include interferon-stimulated genes (ISGs) beyond USP18 as treatment validation markers to confirm pathway activation.

How can USP18 antibodies be employed to investigate its role in viral infections?

USP18 antibodies provide powerful tools for investigating the protein's complex role in viral infections through multiple sophisticated approaches:

Infection Dynamics Analysis: USP18 antibodies can track expression patterns during viral infection time courses, revealing how USP18 regulation correlates with viral replication phases. This is particularly important for viruses like LCMV, VSV, HBV, Sindbis virus, influenza B virus, and HIV, where USP18 has demonstrated significant effects on viral replication .

Biochemical Pathway Mapping: Through co-immunoprecipitation with USP18 antibodies followed by mass spectrometry, researchers can identify viral and host proteins that interact with USP18 during infection. This approach has helped elucidate USP18's inhibitory effect on interferon signaling by demonstrating its binding to IFNAR2 .

Subcellular Localization Studies: Immunofluorescence using USP18 antibodies can reveal how viral infection alters USP18's cellular distribution, providing insights into function. For example, studies have shown that USP18 influences the nuclear translocation of transcription factors p65 and p50 during PRRSV infection .

Chromatin Immunoprecipitation (ChIP): USP18 antibodies used in ChIP assays can determine whether USP18 associates with chromatin during infection, potentially identifying direct gene regulatory roles beyond its enzymatic and interferon-inhibitory functions.

In Vivo Infection Models: Immunohistochemistry with USP18 antibodies in infected tissue samples helps correlate USP18 expression with pathology and viral distribution, particularly valuable in neurotropic infections where USP18 plays critical roles .

What methodological approaches can distinguish between USP18's enzymatic and interferon-regulatory functions?

Distinguishing between USP18's dual functions requires sophisticated experimental designs:

C61A Mutant Comparative Analysis: The C61A mutant lacks isopeptidase activity while maintaining interferon regulation capability. By comparing phenotypes between USP18 knockout, C61A mutant, and wild-type systems, researchers can attribute outcomes to specific functions . Western blotting with USP18 antibodies confirms expression of the mutant protein.

ISG15 Conjugation Profiling: Monitoring global ISG15 conjugation patterns using anti-ISG15 antibodies alongside USP18 detection helps assess USP18's deISGylation function. Increased ISG15 conjugates in USP18 C61A mutant cells but normal interferon signaling confirms separation of functions .

STAT Phosphorylation Kinetics: Measuring phosphorylated STAT1/2 by Western blotting at multiple time points after interferon stimulation can reveal USP18's interferon regulatory function independent of its enzymatic activity . This approach has demonstrated prolonged STAT phosphorylation in USP18-deficient cells.

Domain-Specific Antibodies: Using antibodies that recognize specific domains of USP18 allows monitoring of different functional interactions. Antibodies targeting the C-terminal region might preferentially detect USP18's IFNAR2 binding, while N-terminal antibodies might better assess enzymatic function complexes.

Reconstitution Experiments: Complementing USP18-deficient cells with either wild-type USP18 or the C61A mutant, followed by functional readouts, provides definitive evidence for function separation. This approach requires careful protein expression level monitoring using USP18 antibodies to ensure comparable reconstitution.

How do USP18 expression patterns differ across immune cell populations and what techniques best capture these differences?

USP18 expression varies significantly across immune cell types, with important functional implications:

Expression Profiling Techniques:

• Flow Cytometry: Intracellular staining with USP18 antibodies combined with surface marker panels can quantify USP18 expression in specific immune cell subsets simultaneously . This technique has revealed particularly high USP18 expression in dendritic cells compared to other immune populations .

• Single-Cell RNA-Seq: While not antibody-based, this approach provides comprehensive transcriptional context for USP18 expression patterns and has demonstrated cell type-specific regulation of USP18.

• Immunohistochemistry: Using USP18 antibodies on tissue sections helps visualize expression in anatomical context, revealing tissue-resident immune cell expression patterns .

Cell-Specific Expression Patterns:
Dendritic cells, particularly CD11b+ DCs, express high levels of USP18, which impacts their development and antigen presentation capabilities . USP18 deficiency impairs the generation of CD11b+ DCs both in vitro and in vivo, with fewer cells found in spleen and bone marrow of USP18-deficient mice .

T cells show differential USP18 expression based on activation state and subset. USP18 plays a critical role in Th17 differentiation by inhibiting ubiquitination of the TAK1-TAB complex, thereby suppressing IL-2 production and promoting IL-17 production .

Validation Approaches:
To confirm antibody specificity across different immune cell types, validation using USP18-deficient cells from each population is ideal. Western blotting of sorted cell populations can provide quantitative comparison of expression levels and confirm antibody specificity in each cell type.

What are common sources of background or non-specific signals when using USP18 antibodies, and how can they be mitigated?

Researchers frequently encounter background issues with USP18 antibodies that can be addressed through systematic troubleshooting:

Cross-Reactivity: USP18 belongs to the ubiquitin-specific protease family, which contains many members with structural similarities. To minimize cross-reactivity:

• Use monoclonal antibodies like the D4E7 rabbit mAb that have demonstrated high specificity

• Incorporate USP18 knockout or knockdown controls to identify non-specific bands

• Optimize antibody concentration through careful titration experiments

High Background in Immunostaining: When performing immunofluorescence or immunohistochemistry:

• Extend blocking time (using 5-10% normal serum from the secondary antibody host species)

• Reduce primary antibody concentration or incubation time

• Include detergents (0.1-0.3% Triton X-100) in washing buffers to reduce non-specific binding

• Consider using specialized blocking reagents for tissues with high endogenous biotin or peroxidase activity

Multiple Bands in Western Blots: USP18 may appear as multiple bands due to:

• Post-translational modifications: Run samples with and without interferon stimulation to identify inducible bands

• Degradation products: Add additional protease inhibitors to lysis buffers

• Isoforms: Compare with literature reports of USP18 isoforms and their molecular weights

• Non-specific binding: Use gradient gels (4-12%) to better separate proteins of similar molecular weight

High Background in IP Experiments:

• Pre-clear lysates with protein A/G beads before adding USP18 antibody

• Use USP18 antibodies specifically validated for IP applications, such as the E-5 AC antibody

• Increase the stringency of wash buffers by gradually adjusting salt concentration

How can researchers overcome detection challenges for USP18 in tissues or cells with low expression levels?

Detecting low-abundance USP18 requires specialized technical approaches:

Signal Amplification Methods:

• Implement tyramide signal amplification (TSA) for immunohistochemistry, which can increase sensitivity by 10-100 fold

• Use polymeric detection systems (e.g., EnVision or ImmPRESS) that deliver more enzyme molecules per binding event

• Consider proximity ligation assay (PLA) when studying USP18 interactions, which provides exponential signal amplification

Sample Enrichment Strategies:

• Perform subcellular fractionation to concentrate USP18 from relevant compartments

• Use immunoprecipitation to concentrate USP18 before detection

• For cells with very low expression, consider gentle stimulation with low doses of type I interferons (1-10 IU/ml) to slightly upregulate USP18 without dramatically altering cellular state

Optimized Western Blot Protocol:

• Increase protein loading (up to 50-100μg per lane)

• Use high-sensitivity substrates such as SuperSignal West Femto

• Consider PVDF membranes with smaller pore size (0.2μm) to prevent protein pass-through

• Extend primary antibody incubation to overnight at 4°C with gentle agitation

• Use fluorescent secondary antibodies with infrared detection systems (e.g., Odyssey), which often provide better quantification of low-abundance proteins

Enhanced Microscopy Techniques:

• Employ confocal microscopy with increased laser power and detector gain

• Consider deconvolution microscopy to improve signal-to-noise ratio

• Use super-resolution techniques (STED, STORM) for detailed localization studies of low-abundance USP18

What approaches should be taken when troubleshooting contradictory results from different USP18 antibodies?

Contradictory results between different USP18 antibodies require systematic investigation:

Epitope Mapping Analysis:

• Determine which domains of USP18 are recognized by each antibody

• Antibodies targeting different domains might give different results if:

  • Specific domains are masked by protein interactions

  • Post-translational modifications affect epitope accessibility

  • Protein conformation changes in different experimental conditions

Validation in Knockout/Knockdown Systems:

• Test all antibodies in USP18 knockout or knockdown samples

• A truly specific antibody should show drastically reduced or absent signal in these controls

• Compare results with positive controls (interferon-stimulated samples) to evaluate sensitivity

Cross-Platform Validation Strategy:
When facing contradictory results:

• Compare antibody performance across multiple applications (WB, IHC, IF)

• Validate findings with orthogonal detection methods (mass spectrometry, RNA-seq)

• Correlate protein detection with functional readouts (ISG15 conjugation levels, interferon sensitivity)

Detailed Experimental Conditions Analysis:

• Document exact fixation conditions for each antibody in immunostaining

• Record lysis buffer compositions for protein extraction

• Compare primary antibody incubation conditions (time, temperature, concentration)

• Evaluate different blocking reagents (milk vs. BSA) as they can significantly impact antibody performance

Literature Cross-Reference:

• Review publications using the specific antibodies in question

• Contact authors of key papers for technical advice

• Reference antibody validation resources and databases

How should researchers interpret USP18 expression changes in different disease contexts?

Interpreting USP18 expression changes requires consideration of its dual functions and context-dependent effects:

Viral Infections:
Increased USP18 expression during viral infection represents a complex regulatory mechanism. While upregulation might appear contradictory as it dampens antiviral interferon responses, this actually serves to:

• Prevent excessive inflammation and immunopathology

• Allow sufficient viral replication to stimulate adaptive immunity

• Balance immediate innate response with long-term immunological memory

When interpreting USP18 levels in viral infection studies, researchers should correlate expression with:

• Viral load measurements

• Disease severity markers

• Temporal phase of infection (early vs. late)

• Type I interferon levels and downstream ISG expression

Autoimmune Diseases:
USP18 plays contrasting roles in different autoimmune conditions:

• In autoimmune diabetes, USP18 expression in dendritic cells may promote disease by enabling viral replication that resembles autoantigens

• Conversely, USP18 expression in beta islet cells themselves appears protective, preventing proinflammatory chemokine activity and inhibiting beta cell apoptosis

This cell-type specificity is critical when interpreting USP18 expression data in autoimmune contexts. Researchers should always specify the cellular source of USP18 being measured.

Cancer:
USP18's role in tumorigenesis remains controversial . Elevated USP18 may:

• Negatively impact outcomes by inhibiting interferon's antitumor effects

• Accelerate cell proliferation through various signaling pathways

• Positively impact outcomes by regulating IFN-γ production and maintaining antigen-specific CTLs in the tumor microenvironment

When interpreting USP18 expression in cancer tissues, researchers should correlate with:

• Tumor type and stage

• Immune infiltration profiles

• Patient treatment history, particularly prior exposure to interferons

• Survival outcomes and treatment response

What experimental approaches can determine whether USP18 serves a protective or pathogenic role in specific disease models?

Establishing USP18's functional role requires sophisticated experimental designs:

Conditional and Cell-Type Specific Knockout Models:
Because USP18 functions differ by cell type, conditional knockout approaches provide crucial insights:

• USP18 deletion in dendritic cells versus beta islet cells has opposite effects on diabetes development

• Compare phenotypes between global USP18 knockout and cell-type specific deletions

• Use Cre-lox systems under cell-type specific promoters to achieve targeted deletion

Separation of Function Mutants:
The C61A mutant allows dissection of isopeptidase activity from interferon regulation:

• Compare disease progression in wild-type, USP18 knockout, and C61A mutant animals

• This approach revealed that C61A mutants lack brain abnormalities seen in complete knockouts

• Establish whether disease modulation requires enzymatic activity or interferon regulation

Temporal Intervention Studies:
Using inducible systems or timed interventions determines when USP18 functions are critical:

• Administer interferon-blocking antibodies at different disease stages

• Use tetracycline-inducible USP18 expression systems

• Apply pharmaceutical inhibitors of USP18 at defined disease timepoints

Correlated Multi-Parameter Analysis:
Integrating multiple readouts provides mechanistic understanding:

• Measure USP18 expression/activity

• Assess ISG15 conjugation levels

• Quantify interferon pathway activation (pSTAT1/2)

• Evaluate disease-specific parameters

• Monitor immune cell populations and their activation states

Therapeutic Intervention Testing:
Testing USP18 modulation as treatment provides functional insights:

• Administer USP18 inhibitors or activators to disease models

• Use USP18-targeting siRNA/shRNA delivered to specific tissues

• Test interferon therapy in USP18-deficient versus wild-type backgrounds

How can USP18 antibodies be employed in developing potential therapeutic approaches for autoimmune diseases or viral infections?

USP18 antibodies serve critical roles in therapeutic development pipelines:

Target Validation Studies:

• USP18 antibodies in immunohistochemistry confirm protein expression in disease-relevant tissues

• Quantitative Western blotting with USP18 antibodies correlates expression levels with disease severity

• Co-localization studies identify which specific cell populations express USP18 in disease states

Therapeutic Antibody Development:
For developing therapeutic antibodies against USP18:

• Use existing research-grade antibodies to identify accessible epitopes

• Screen therapeutic candidates against these epitopes

• Employ competition assays with validated USP18 antibodies to confirm binding

• Use USP18 antibodies as positive controls in functional assays

Pharmacodynamic Biomarker Analysis:
USP18 antibodies assess treatment efficacy in clinical trials:

• Measure changes in USP18 expression levels after treatment

• Quantify downstream pathway effects (ISGylation, STAT phosphorylation)

• Correlate molecular changes with clinical outcomes

Companion Diagnostic Development:
USP18 antibodies can help stratify patients:

• Identify patients with abnormal USP18 expression who might benefit from targeted therapies

• Develop immunohistochemistry protocols using validated USP18 antibodies for diagnostic use

• Create standardized scoring systems for USP18 expression in patient samples

Drug Mechanism Studies:
For small molecule USP18 inhibitors/modulators:

• Confirm target engagement using cellular thermal shift assays (CETSA) with USP18 antibodies

• Assess drug effects on USP18 protein stability and turnover

• Evaluate changes in USP18 protein interactions using co-immunoprecipitation

What emerging technologies might enhance the utility of USP18 antibodies in research?

Several cutting-edge technologies promise to expand USP18 antibody applications:

Proximity-Based Labeling Technologies:

• BioID or APEX2 fusions with USP18 combined with specific antibodies can map the USP18 interactome in living cells

• TurboID systems provide faster labeling kinetics, allowing temporal mapping of USP18 interactions during interferon responses

• Split-BioID approaches could reveal conditional interactions dependent on USP18's activation state

Single-Cell Protein Analysis:

• Mass cytometry (CyTOF) with metal-conjugated USP18 antibodies enables high-dimensional analysis of USP18 expression alongside dozens of other proteins at single-cell resolution

• Imaging mass cytometry combines tissue context with single-cell USP18 quantification

• CODEX multiplexed imaging allows visualization of USP18 alongside >40 other proteins in tissue sections

Intrabody Development:

• Converting validated USP18 antibodies into intrabodies (intracellular antibodies) enables real-time visualization of USP18 in living cells

• nanobody-based approaches provide smaller probes with better access to restricted cellular compartments

• CRISPR epitope tagging combined with anti-tag antibodies offers endogenous USP18 tracking without overexpression artifacts

Spatial Transcriptomics Integration:

• Combining USP18 immunohistochemistry with spatial transcriptomics provides cellular context for USP18 function

• Multiplexed error-robust FISH (MERFISH) with USP18 protein detection reveals relationship between transcription and protein levels

• visium spatial gene expression platform permits correlation of USP18 protein localization with global gene expression patterns

Antibody Engineering Approaches:

• Bispecific antibodies targeting USP18 and interacting partners

• Split-fluorescent protein complementation using anti-USP18 antibody fragments for interaction studies

• nanobody-based biosensors to detect USP18 conformational changes upon binding partners or substrates

How might comparative analysis of USP18 across species inform our understanding of its conserved functions?

Cross-species USP18 analysis provides evolutionary and functional insights:

Evolutionary Conservation Mapping:

• USP18 antibodies with cross-species reactivity allow direct comparison of expression patterns

• Western blot analysis of USP18 across species reveals size variations that may indicate functional adaptations

• Immunoprecipitation followed by mass spectrometry can identify species-specific interaction partners

Comparative Functional Analysis:
Human USP18 and mouse Usp18 show both similarities and differences:

These differences can be explored using:

• Cross-species protein complementation experiments

• Domain-swapping between human and mouse USP18 followed by functional assays

• Analysis of species-specific post-translational modifications using specific antibodies

Infection Model Comparative Studies:

• Different species show varying USP18 responses to viral infections

• USP18 antibodies can track expression kinetics across species after identical stimuli

• Comparing USP18 regulation between natural hosts and experimental models of viral infection may explain differences in pathogenesis

Therapeutic Relevance Assessment:

• Cross-reactive USP18 antibodies allow testing of therapeutic concepts in animal models

• Identification of species-specific differences informs translation of preclinical findings

• Epitope conservation analysis helps predict antibody cross-reactivity for therapeutic development

What are the most promising approaches for developing specific inhibitors or modulators of USP18 function?

Development of USP18 modulators represents an exciting frontier with several promising approaches:

Structure-Guided Drug Design:

• Crystal structures of USP18 catalytic domain inform rational design of inhibitors

• USP18 antibodies can be used in co-crystallization studies to stabilize specific conformations

• Fragment-based screening against purified USP18 domains identifies starting points for inhibitor development

Differential Targeting Strategies:
Given USP18's dual functions, selective targeting approaches include:

• Catalytic site inhibitors that block deISGylase activity while preserving interferon regulation

• Protein-protein interaction disruptors that prevent IFNAR2 binding without affecting enzymatic function

• Allosteric modulators that alter USP18 conformation and function without directly blocking active sites

Activity-Based Probes:

• Development of USP18-specific activity-based probes

• USP18 antibodies validate probe specificity in complex biological samples

• These probes serve both as research tools and as prototypes for inhibitor development

Targeted Protein Degradation:

• PROTAC (Proteolysis-Targeting Chimera) approach linking USP18-binding molecules to E3 ligase recruiters

• Antibody-PROTAC conjugates combining the specificity of USP18 antibodies with degradation technology

• Degradation tag systems for rapid, reversible control of USP18 levels in research settings

Therapeutic Antibody Approaches:

• Developing antibodies that selectively block specific USP18 functions

• Intracellular antibody delivery systems to target USP18 within cells

• Antibody-drug conjugates to deliver USP18 modulators to specific cell populations

Validation Methods:
For all approaches, validation requires:

• Target engagement confirmation (cellular thermal shift assays with USP18 antibodies)

• Functional readouts (ISG15 conjugation levels, interferon signaling)

• Selectivity profiling against other USPs

• Cellular efficacy assessment

• In vivo efficacy in disease models

Table 1: Comparison of USP18 Targeting Approaches