STUB1 Antibody, FITC conjugated

What is STUB1 and why is it significant in immunological research?

STUB1/CHIP is an E3 ubiquitin ligase that plays critical roles in protein quality control and immune regulation. Recent research demonstrates that STUB1 contributes significantly to the Th17/Treg cell imbalance through non-degradative ubiquitination of the aryl hydrocarbon receptor (AHR) . This protein has emerged as particularly important in autoimmune disease research, as its expression is notably altered in conditions like rheumatoid arthritis (RA), where it is upregulated in Th17 cells and downregulated in Treg cells from RA patients compared to healthy controls . The protein's ability to modify immune cell differentiation through ubiquitination pathways makes it a valuable target for both basic and translational research.

What cellular compartments typically show STUB1 expression and how might this influence antibody selection?

STUB1 functions primarily in the cytoplasm as part of protein quality control mechanisms, but its activity extends to nuclear proteins as well. Based on its interactions with AHR (which translocates between cytoplasm and nucleus) and AGO2 (found in both compartments), researchers should consider membrane permeabilization protocols when using FITC-conjugated STUB1 antibodies for intracellular staining . For flow cytometry applications, standard permeabilization methods using saponin or methanol are typically sufficient, but optimization may be required depending on cell type and fixation method. When designing immunofluorescence experiments, consider that STUB1's distribution may change under different cellular conditions, particularly in activated versus resting immune cells.

What are the optimal protocols for using FITC-conjugated STUB1 antibodies in flow cytometry?

For flow cytometric analysis of STUB1 in T cell populations, particularly when examining Th17/Treg imbalance, researchers should follow these methodological steps:

• Isolate CD4+ T cells using magnetic beads or flow sorting

• Fix cells with 4% paraformaldehyde for 15-20 minutes at room temperature

• Permeabilize with 0.1% Triton X-100 or commercial permeabilization buffer

• Block with 1-5% BSA for 30 minutes

• Incubate with FITC-conjugated STUB1 antibody (typically 1:100-1:500 dilution)

• For co-staining Th17/Treg populations, include antibodies against IL-17 and Foxp3

• Analyze using appropriate compensation controls for multi-color flow cytometry

Considering the differential expression of STUB1 in Th17 versus Treg cells in RA patients, it's advisable to optimize antibody concentration for each cell type . Additionally, when examining STUB1's role in T cell differentiation, consider time-course experiments that capture its dynamic regulation during polarization processes.

How can I design experiments to explore STUB1's role in Th17/Treg imbalance using FITC-conjugated antibodies?

Based on the research findings, a comprehensive experimental approach would include:

• Cell isolation and culture: Isolate CD4+ T cells from peripheral blood of both healthy donors and RA patients.

• Manipulation of STUB1 expression: Use lentiviral vectors (LV-STUB1 for overexpression, LV-sh-STUB1 for knockdown) to modify STUB1 levels.

• Polarization conditions: Culture cells under Th17 or Treg polarizing conditions with anti-CD3/CD28 stimulation.

• Flow cytometry analysis: Use FITC-conjugated STUB1 antibody alongside markers for Th17 (CD4+IL-17+) and Treg (CD25+Foxp3+) cells.

• Cytokine profiling: Measure IL-17A, IL-6, TNF-α (Th17-associated) and IL-10, TGF-β (Treg-associated) in culture supernatants.

• qRT-PCR: Analyze expression of RORγt, IL-17A, and Foxp3 to correlate with protein-level findings.

This experimental design allows researchers to directly correlate STUB1 expression levels with T cell differentiation outcomes and cytokine production . The FITC-conjugated antibody enables direct visualization of STUB1 in these different cellular contexts without requiring additional detection steps.

What controls are essential when using FITC-conjugated STUB1 antibodies?

For rigorous experimental design with FITC-conjugated STUB1 antibodies, include these essential controls:

• Isotype control: Use a FITC-conjugated antibody of the same isotype (typically rabbit polyclonal for STUB1 antibodies) to assess non-specific binding .

• Fluorescence minus one (FMO) control: Include all antibodies in your panel except the STUB1-FITC to establish proper gating strategies.

• STUB1 knockdown/knockout samples: When available, include samples with confirmed STUB1 suppression to validate antibody specificity.

• Blocking peptide control: Pre-incubate antibody with the immunizing peptide before staining to confirm binding specificity.

• Unstained and single-stained controls: Essential for establishing proper compensation in multicolor flow cytometry.

These controls are particularly important when studying STUB1 in the context of Th17/Treg balance, where subtle differences in expression levels may have functional significance .

How can FITC-conjugated STUB1 antibodies be used to investigate STUB1-mediated ubiquitination of AHR?

To investigate STUB1's non-degradative ubiquitination of AHR using FITC-conjugated STUB1 antibodies, researchers can implement this advanced protocol:

• Co-immunoprecipitation setup:

  • Perform co-IP using anti-AHR antibodies

  • Probe for STUB1 using FITC-conjugated antibodies in fluorescent Western blotting

  • Probe for ubiquitination using anti-ubiquitin antibodies

• Confocal microscopy visualization:

  • Perform immunofluorescence with FITC-conjugated STUB1 antibodies

  • Co-stain with AHR-specific antibodies and ubiquitin antibodies

  • Analyze colocalization with high-resolution confocal microscopy

• Flow cytometry-based protein interaction analysis:

  • Use proximity ligation assay (PLA) techniques with FITC-STUB1 and AHR antibodies

  • Quantify interaction events per cell using flow cytometry

The research findings show that STUB1 promotes non-degradative K63-linked ubiquitination of AHR, rather than the proteasome-targeting K48-linked ubiquitination it performs on other substrates like AGO2 . This methodological approach allows researchers to distinguish between these different ubiquitination patterns and their functional consequences.

What are the considerations for investigating STUB1's role in multiple signaling pathways simultaneously?

STUB1 participates in multiple signaling pathways, including T cell differentiation through AHR and RNA interference through AGO2 ubiquitination. When designing multiplex experiments to examine these pathways simultaneously, consider:

• Antibody panel design: Carefully select fluorophores with minimal spectral overlap when combining FITC-STUB1 with antibodies against AHR, AGO2, and other pathway components.

• Sequential immunoprecipitation approach:

  • First IP: Pull down STUB1 complexes

  • Elution and second IP: Separate into AHR-associated and AGO2-associated fractions

  • Analysis: Compare ubiquitination patterns and associated proteins

• Pathway inhibition strategy: Use specific inhibitors of AHR (e.g., CH223191) or RNA interference components to dissect STUB1's differential roles.

• Time-course experiments: Monitor STUB1 associations with different pathways at various timepoints after T cell activation or viral challenge.

This approach acknowledges STUB1's context-dependent functions, where it mediates non-degradative ubiquitination in the AHR pathway but promotes proteasomal degradation in the AGO2 pathway .

How can FITC-conjugated STUB1 antibodies be used to investigate its role in antiviral RNAi mechanisms?

Based on the finding that STUB1 regulates antiviral RNAi through AGO2 ubiquitination and degradation , researchers can use FITC-conjugated STUB1 antibodies to:

• Virus infection models:

  • Infect cells with model viruses (e.g., VSV, influenza)

  • Track STUB1 localization using FITC-conjugated antibodies

  • Correlate with AGO2 levels and viral replication

• Subcellular fractionation analysis:

  • Separate cytoplasmic and nuclear fractions

  • Quantify STUB1 redistribution during viral infection

  • Correlate with formation of RNA-induced silencing complexes

• Live-cell imaging:

  • Use FITC-STUB1 antibodies for immunofluorescence in fixed timepoints

  • Track colocalization with AGO2 and viral components

  • Quantify formation of RNA processing bodies and stress granules

This methodological approach enables researchers to visualize the dynamic relationship between STUB1 and components of the RNAi machinery during viral infection, particularly focusing on how STUB1-mediated ubiquitination affects AGO2 stability and function in antiviral responses .

What strategies can overcome potential epitope masking when detecting STUB1 in protein complexes?

STUB1 functions within protein complexes, particularly with chaperones and its ubiquitination targets, which may cause epitope masking. Consider these methodological refinements:

• Epitope retrieval optimization:

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Try different buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Optimize incubation times for each cell type

• Detergent panel testing:

  • Compare mild (0.1% Triton X-100) vs. stronger detergents (1% SDS)

  • Consider digitonin for selective membrane permeabilization

  • Test NP-40 alternatives for preservation of protein complexes

• Denaturing vs. native conditions:

  • For complete protein complex disruption, use denaturing conditions

  • For preservation of physiological interactions, use native conditions

The choice of method depends on your experimental question. When studying STUB1's interaction with AHR, milder conditions may preserve the interaction for co-detection . For detecting total STUB1 levels regardless of binding partners, more stringent conditions may be necessary.

How can I optimize signal-to-noise ratio when using FITC-conjugated STUB1 antibodies in tissues with high autofluorescence?

When working with tissues that exhibit high autofluorescence in the FITC channel (particularly synovial tissues from RA patients), consider these optimization strategies:

• Autofluorescence reduction techniques:

  • Sudan Black B treatment (0.1-0.3% in 70% ethanol)

  • Copper sulfate treatment (1mM CuSO4 in 50mM ammonium acetate)

  • TrueBlack® or similar commercial autofluorescence quenchers

• Detection optimization:

  • Consider longer wavelength alternatives to FITC if possible

  • Use spectral unmixing on confocal platforms

  • Implement linear unmixing algorithms during image analysis

• Alternative amplification methods:

  • Biotin-streptavidin amplification systems

  • Tyramide signal amplification

  • Quantum dot secondary detection

• Microscopy settings optimization:

  • Narrow bandpass filters to exclude autofluorescence wavelengths

  • Reduced exposure time with increased antibody concentration

  • Confocal pinhole adjustment to minimize out-of-focus signal

These approaches are particularly relevant when examining STUB1 expression in synovial tissues from RA patients, where visualization of the differential expression between Th17 and Treg cells requires optimal signal-to-noise ratios .

What are the best practices for quantifying STUB1 expression differences between Th17 and Treg cells?

Based on the findings that STUB1 is differentially expressed in Th17 versus Treg cells from RA patients , these analytical approaches are recommended:

• Flow cytometry quantification:

  • Report median fluorescence intensity (MFI) rather than mean

  • Calculate MFI ratio of STUB1-FITC to isotype control

  • Use standardized beads for day-to-day calibration

• Statistical considerations:

  • Paired analysis for Th17 and Treg cells from the same donor

  • Non-parametric tests if distribution normality cannot be confirmed

  • Consider fold-change compared to healthy controls

• Multiparameter correlation:

  • Correlate STUB1 levels with functional cytokine production

  • Analyze relationship between STUB1 and transcription factors (RORγt, Foxp3)

  • Examine correlation with clinical parameters in RA patients

This quantification approach allows for robust comparison between patient cohorts and experimental conditions, facilitating mechanistic understanding of how STUB1 contributes to immune dysregulation in autoimmune diseases .

How should researchers interpret contradictory data regarding STUB1's ubiquitination targets?

The research findings present an interesting dichotomy in STUB1's ubiquitination activity—non-degradative K63-linked ubiquitination of AHR versus degradative K48-linked ubiquitination of AGO2 . When encountering seemingly contradictory data about STUB1's effects on different targets, consider these analytical frameworks:

• Context-dependent activity analysis:

  • Compare cellular contexts (T cells vs. other cell types)

  • Examine influence of activation state on ubiquitination patterns

  • Consider tissue-specific cofactors that may direct STUB1 activity

• Ubiquitin linkage determination:

  • Use linkage-specific antibodies (anti-K48 vs. anti-K63)

  • Employ mass spectrometry to identify ubiquitination sites

  • Perform mutational analysis of potential ubiquitination residues

• Temporal dynamics consideration:

  • Analyze early vs. late timepoints after stimulation

  • Examine ubiquitination patterns during different phases of immune response

  • Consider kinetic differences between degradative and non-degradative ubiquitination

• Integration with functional readouts:

  • Correlate ubiquitination patterns with functional outcomes (T cell differentiation, RNAi efficiency)

  • Use inhibitors of specific ubiquitin pathways to dissect mechanisms

  • Examine downstream signaling consequences of different ubiquitination patterns

This analytical approach acknowledges that E3 ligases like STUB1 often have context-dependent functions, allowing researchers to reconcile seemingly contradictory findings about its roles in different biological processes .

How might FITC-conjugated STUB1 antibodies be used to explore its therapeutic potential in autoimmune diseases?

Given STUB1's role in promoting Th17/Treg imbalance in RA , FITC-conjugated antibodies could facilitate translational research through:

• High-throughput screening applications:

  • Screen compounds that modulate STUB1 expression or activity

  • Use flow cytometry with FITC-STUB1 antibodies as a primary readout

  • Correlate with T cell differentiation outcomes

• Patient stratification approaches:

  • Analyze STUB1 expression patterns in RA patient subgroups

  • Correlate with treatment responses to biological therapies

  • Develop predictive biomarker panels including STUB1

• Ex vivo therapeutic response modeling:

  • Isolate patient cells and treat with candidate compounds

  • Monitor STUB1 levels and localization using FITC-conjugated antibodies

  • Assess normalization of Th17/Treg balance after treatment

• In vivo treatment monitoring:

  • Analyze STUB1 expression in patient samples before and after treatment

  • Correlate changes with clinical response measures

  • Identify early molecular indicators of treatment efficacy

These approaches leverage FITC-conjugated STUB1 antibodies as tools for developing personalized medicine approaches for autoimmune diseases, where restoring proper Th17/Treg balance represents a promising therapeutic strategy .

What methodological approaches can investigate the relationship between STUB1 and other E3 ligases in immune regulation?

To explore how STUB1 functions within the broader network of E3 ligases that regulate immune responses, researchers can employ these methodological approaches:

• Competitive binding studies:

  • Use FITC-STUB1 antibodies alongside antibodies for other E3 ligases

  • Analyze competition for shared substrates (e.g., AHR)

  • Quantify binding preferences under different cellular conditions

• E3 ligase activity profiling:

  • Develop activity-based probes for multiple E3 ligases

  • Combine with FITC-STUB1 antibodies to correlate expression and activity

  • Create activity maps under different immune conditions

• Genetic interaction analysis:

  • Perform CRISPR screens targeting multiple E3 ligases

  • Use FITC-STUB1 antibodies to assess compensatory expression changes

  • Identify synthetic lethal or redundant relationships

• Pathway integration mapping:

  • Use systems biology approaches to map E3 ligase networks

  • Position STUB1 within larger ubiquitination cascades

  • Identify critical nodes for therapeutic intervention

This comprehensive approach recognizes that STUB1 operates within a complex network of ubiquitination machinery, potentially interacting with or compensating for other E3 ligases in regulating immune cell differentiation and function .