STUB1 Antibody

What is STUB1 and why is it a significant research target?

STUB1 (also known as CHIP - Carboxy terminus of Hsp70-interacting protein) is a RING-type E3 ubiquitin ligase that plays crucial roles in protein quality control and cellular homeostasis. Its significance stems from its function as a negative regulator of several important proteins through ubiquitination and subsequent proteasomal degradation. Research has shown that STUB1 regulates critical transcription factors such as RUNX1 and its leukemogenic fusion protein RUNX1-RUNX1T1 . Additionally, STUB1 has been implicated in cell senescence processes through its interaction with circadian rhythm proteins like BMAL1 . The multifaceted roles of STUB1 in protein degradation pathways make it an important target for research in areas including cancer biology, neurodegeneration, and aging-related diseases.

What types of STUB1 antibodies are available for research applications?

Based on current research tools, STUB1 antibodies are available in several formats optimized for different experimental applications:

• Polyclonal antibodies: Such as rabbit polyclonal antibodies (e.g., ab2917) that recognize epitopes within the C-terminal region (aa 200-250) of human STUB1

• Monoclonal antibodies: Offering higher specificity for particular epitopes

• Species-specific antibodies: Available for human, mouse, and other model organisms

• Application-specific formulations: Optimized for Western blotting, immunoprecipitation, immunofluorescence, or chromatin immunoprecipitation

The choice of antibody depends on the specific research question, experimental technique, and model system being used. Researchers should consider the validation status of the antibody for their specific application and species of interest.

What are the appropriate sample types for STUB1 antibody applications?

STUB1 antibodies have been validated for use with various biological samples including:

• Cell lysates from both endogenous expression systems and transfected cells overexpressing STUB1

• Tissue extracts, particularly from brain, muscle, and cancer tissues

• Nuclear and cytoplasmic fractions (important given STUB1's differential activity in these compartments)

• Immunoprecipitated protein complexes for studying STUB1 interactions

For example, research has successfully utilized STUB1 antibodies to detect the protein in mouse brain samples, human-derived cell lines like K562 and Kasumi-1, and in transfected cell lysates from 293T cells . When selecting samples, researchers should consider the expression level of STUB1 in their system of interest, as levels may vary significantly across different cell and tissue types.

How should Western blot protocols be optimized for STUB1 detection?

For optimal STUB1 detection via Western blotting, the following methodological considerations should be implemented:

• Sample preparation: Total protein loading of 20-30 μg is typically sufficient for detecting endogenous STUB1 in most cell types. Use RIPA or NP-40 lysis buffers supplemented with protease inhibitors.

• Electrophoresis conditions: Use 10-12% SDS-PAGE gels for optimal separation, as STUB1 has a predicted molecular weight of approximately 35 kDa .

• Transfer parameters: Semi-dry or wet transfer methods are suitable, with PVDF membranes often providing better results than nitrocellulose for STUB1 detection.

• Blocking conditions: 5% non-fat dry milk in TBST is typically effective for blocking non-specific binding .

• Antibody dilutions: Primary antibody dilutions of 1:1000 are typically effective, though this may vary by specific antibody and application .

• Detection method: While both chemiluminescent and fluorescent detection methods work well, enhanced chemiluminescence provides good sensitivity for detecting physiological levels of STUB1.

• Controls: Include both positive controls (e.g., STUB1-overexpressing cells) and negative controls (e.g., STUB1-knockout or depleted samples) to validate specificity.

How can researchers distinguish between free STUB1 and protein-bound complexes in experimental systems?

Distinguishing between free STUB1 and its protein complexes requires specialized experimental approaches:

• Size exclusion chromatography combined with Western blotting: This allows separation of protein complexes based on molecular weight before antibody detection.

• Co-immunoprecipitation with differential detergent conditions: Varying detergent strengths can help dissociate weak vs. strong protein interactions before STUB1 antibody immunoprecipitation.

• Proximity ligation assays: These can detect STUB1 interactions with specific partners (e.g., BMAL1 or RUNX1) in situ with high specificity.

• Subcellular fractionation: As demonstrated in research, STUB1-induced ubiquitination of RUNX1 occurs predominantly in the nucleus . Therefore, separating nuclear and cytoplasmic fractions before immunoprecipitation can help identify compartment-specific STUB1 complexes.

• Native PAGE vs. SDS-PAGE: Native conditions preserve protein complexes while denaturing conditions reveal total STUB1 levels.

When investigating potential interaction partners, researchers should consider that the U-box domain of STUB1 has been identified as critical for interactions with targets like BMAL1 . Additionally, chaperone proteins such as Hsp70 and Hsp90 often mediate STUB1's interactions with its substrates, as evidenced by the reduced efficiency of the chaperone-binding deficient STUB1-K30A mutant in ubiquitinating RUNX1 .

What approaches can resolve contradictory findings about STUB1 substrate specificity in different cellular contexts?

Researchers facing contradictory results regarding STUB1 substrate specificity should consider implementing these methodological approaches:

• Cell type-specific expression analysis: Perform comparative analysis of STUB1 expression levels across different cell types. For example, STUB1 expression has been shown to vary significantly across leukemia cell lines, with HEL cells showing the lowest expression while other cell lines exhibited higher levels .

• Chaperone dependency assessment: Examine the requirement for specific chaperones (Hsp70/Hsp90) in different cellular contexts by using:

  • Chaperone inhibitors (e.g., 17-AAG for Hsp90)

  • Chaperone-binding deficient STUB1 mutants (e.g., STUB1-K30A)

  • Co-immunoprecipitation to assess chaperone recruitment differences

• Parallel substrate validation: When conflicting results arise regarding a potential STUB1 substrate:

  • Compare ubiquitination patterns using both overexpression and endogenous systems

  • Validate with multiple antibodies targeting different STUB1 epitopes

  • Perform domain mapping to identify critical interaction regions

• Post-translational modification analysis: STUB1 activity can be regulated by its own post-translational modifications. Mass spectrometry analysis of STUB1 from different cellular contexts can reveal modifications that might explain differential substrate targeting.

• Competitive binding assays: When multiple substrates are present, perform competition assays to determine preferential targeting under physiological conditions.

How can researchers effectively monitor STUB1-mediated ubiquitination dynamics in real-time?

Monitoring STUB1-mediated ubiquitination dynamics requires sophisticated approaches beyond standard fixed-timepoint assays:

• FRET-based ubiquitination sensors: These can be constructed using fluorescently-tagged ubiquitin and substrate proteins to monitor real-time ubiquitination events in living cells.

• Pulse-chase experiments with cycloheximide: This approach, as used in studies of RUNX1 stability, allows tracking of protein degradation rates in the presence or absence of STUB1 . Results showed that STUB1 depletion significantly extended the half-life of RUNX1 protein.

• Ubiquitin chain linkage-specific antibodies: These allow discrimination between different ubiquitin chain types (K48 vs. K63 linkages) that may dictate different fates for STUB1 substrates.

• Proteasome inhibition time-course: Treatment with inhibitors like MG132 at different time points can help distinguish between ubiquitination leading to degradation versus non-degradative signaling.

• Advanced microscopy techniques:

  • Fluorescence recovery after photobleaching (FRAP) to monitor turnover rates

  • Single-molecule tracking to follow individual ubiquitination events

  • Photoactivatable fluorescent proteins fused to STUB1 substrates

When implementing these approaches, it's important to consider that STUB1-mediated ubiquitination can have outcomes beyond proteasomal degradation, including alterations in subcellular localization. For instance, studies have shown that STUB1-mediated ubiquitination of RUNX1 promotes its nuclear export, contributing to reduced transcriptional activity .

What are the critical quality control steps for validating STUB1 antibodies before experimental use?

Rigorous validation of STUB1 antibodies is essential for reliable experimental outcomes. Researchers should implement these quality control steps:

• Specificity validation:

  • Test antibody against STUB1-depleted samples (using CRISPR/Cas9 or siRNA knockdown)

  • Compare detection patterns with multiple antibodies targeting different STUB1 epitopes

  • Perform peptide competition assays with the immunizing peptide

• Cross-reactivity assessment:

  • Evaluate potential cross-reactivity with other U-box domain proteins

  • Test across multiple species if planning cross-species experiments

  • Validate in samples with varying STUB1 expression levels

• Application-specific validation:

  • For Western blotting: Confirm the correct molecular weight (35 kDa) and perform gradient dilutions to establish detection limits

  • For immunoprecipitation: Validate with both overexpressed and endogenous STUB1

  • For immunofluorescence: Compare patterns with subcellular markers and test fixation methods

• Lot-to-lot consistency:

  • Maintain reference samples for comparison across antibody lots

  • Document key performance metrics for longitudinal comparison

• Functional validation:

  • Confirm that antibody can detect changes in STUB1 levels following established stimuli

  • Verify ability to distinguish between wild-type STUB1 and functional mutants

How should researchers design experiments to investigate STUB1's dual roles in nuclear and cytoplasmic compartments?

STUB1 exhibits distinct activities in nuclear and cytoplasmic compartments, requiring careful experimental design to dissect these functions:

• Subcellular fractionation protocols:

  • Optimize nuclear-cytoplasmic fractionation with minimal cross-contamination

  • Validate fractionation quality using compartment-specific markers

  • Consider using multiple fractionation methods to confirm findings

• Compartment-specific STUB1 mutants:

  • Design nuclear localization signal (NLS) or nuclear export signal (NES) fusion constructs

  • Create tethering constructs to restrict STUB1 to specific compartments

  • Validate localization using both immunofluorescence and biochemical fractionation

• Time-course analyses:

  • Monitor dynamic shuttling of STUB1 and its substrates between compartments

  • Combine with inhibitors of nuclear transport (e.g., leptomycin B)

  • Use photoactivatable constructs to track movement in real-time

• Substrate-specific considerations:

  • For nuclear substrates like RUNX1, design experiments that can distinguish between degradation and nuclear export

  • For cytoplasmic substrates, consider the role of chaperones in recruitment

• Analytical approaches:

  • Quantify the relative ubiquitination efficiency in different compartments

  • Compare substrate half-lives in different compartments

  • Assess how ubiquitination affects substrate localization and function

What strategies can effectively differentiate between direct and indirect effects of STUB1 on target proteins?

Distinguishing direct from indirect effects of STUB1 on target proteins requires multilayered experimental approaches:

• In vitro ubiquitination assays:

  • Reconstitute ubiquitination using purified components (E1, E2, STUB1, and substrate)

  • Compare reaction efficiency with and without chaperone proteins

  • Use catalytically inactive STUB1 (H260Q) as negative control

• Domain mapping and interaction studies:

  • Identify direct binding interfaces between STUB1 and putative substrates

  • Use truncation mutants to define minimal interaction domains, as demonstrated with the U-box domain of STUB1 binding to BMAL1

  • Employ peptide arrays to pinpoint specific interaction motifs

• Temporal analyses:

  • Use rapid induction systems (e.g., auxin-inducible degron) to distinguish immediate vs. delayed effects

  • Combine with protein synthesis inhibitors like cycloheximide to eliminate secondary effects requiring new protein synthesis

• Multi-omics approaches:

  • Compare ubiquitinome, proteome, and interactome changes following STUB1 manipulation

  • Use quantitative proteomics to identify proteins with altered stability

  • Apply network analysis to distinguish direct substrates from downstream effectors

• Rescue experiments:

  • Test whether reintroduction of wild-type vs. mutant STUB1 reverses observed phenotypes

  • Design substrate mutants resistant to STUB1-mediated ubiquitination to confirm specificity

How can researchers address inconsistent STUB1 antibody detection in immunoprecipitation experiments?

Inconsistent STUB1 antibody performance in immunoprecipitation can be addressed through systematic troubleshooting:

• Antibody-substrate binding interference:

  • The epitope recognized by the antibody may overlap with substrate binding regions

  • Solution: Use multiple antibodies targeting different STUB1 epitopes

  • Alternatively, epitope-tag STUB1 at termini unlikely to interfere with function

• Complex stabilization challenges:

  • STUB1-substrate interactions may be transient or destabilized during lysis

  • Solution: Use crosslinking agents prior to lysis (e.g., DSP or formaldehyde)

  • Add proteasome inhibitors (MG132) to stabilize ubiquitinated intermediates

• Buffer optimization:

  • Test multiple lysis buffers with varying detergent types and strengths

  • Include ATP and/or ATPase inhibitors to stabilize chaperone-mediated interactions

  • Add deubiquitinase inhibitors (e.g., N-ethylmaleimide) to preserve ubiquitination

• Technical optimization:

  • Adjust antibody-to-bead ratio (typical range 2-10 μg antibody per 25-50 μl beads)

  • Test different incubation times and temperatures for antibody-antigen binding

  • Consider protein A vs. protein G beads based on antibody isotype

• Species-specific considerations:

  • Ensure compatibility between the antibody source species and the secondary reagents

  • For tissue samples, block endogenous immunoglobulins with species-specific blocking reagents

What are effective strategies for optimizing antibody-based detection of STUB1-substrate complexes in different experimental systems?

Optimizing detection of STUB1-substrate complexes requires consideration of several critical factors:

• Complex-stabilizing conditions:

  • Use mild lysis conditions to preserve protein-protein interactions

  • Include ATP (1-5 mM) to maintain chaperone-mediated interactions

  • Add zinc chelators cautiously, as the U-box domain is zinc-dependent but excessive chelation may disrupt structure

• Sequential immunoprecipitation approaches:

  • First immunoprecipitate the substrate, then probe for STUB1

  • Alternatively, immunoprecipitate STUB1 and probe for substrate

  • Compare results from both approaches to confirm interaction

• System-specific considerations:

  • For cell culture: Synchronize cells if the interaction is cell-cycle dependent

  • For tissue samples: Optimize extraction methods to preserve native complexes

  • For recombinant systems: Include appropriate chaperones (Hsp70/Hsp90)

• Detection enhancement strategies:

  • Amplify signal using biotin-streptavidin systems for low-abundance complexes

  • Use proximity ligation assays for detecting interactions with spatial context

  • Consider mass spectrometry for unbiased complex identification

• Controls and validations:

  • Include competition with recombinant proteins or peptides

  • Use STUB1 mutants with disrupted substrate interaction domains

  • Compare results across multiple cell types with varying STUB1/substrate ratios

Researchers should note that STUB1-substrate interactions often depend on post-translational modifications of the substrate, and these modifications may vary across experimental systems. For example, the interaction between STUB1 and RUNX1-RUNX1T1 in leukemia cells may be regulated differently than in non-hematopoietic cells .

How can STUB1 antibodies be leveraged to develop therapeutic strategies for RUNX1-RUNX1T1 leukemia?

Research has revealed promising therapeutic implications for targeting STUB1 in RUNX1-RUNX1T1 leukemia, with several investigational approaches:

• STUB1 activation as therapeutic strategy:

  • Research has demonstrated that STUB1 overexpression specifically inhibits the growth of RUNX1-RUNX1T1 leukemia cells while showing minimal effect on non-RUNX1-RUNX1T1 leukemia cell lines and normal human cord blood cells

  • Growth inhibition occurs through increased apoptosis and cell cycle arrest in these leukemia cells

• Antibody-based screening platforms:

  • Develop high-throughput screening assays using STUB1 antibodies to identify:

    • Small molecules that enhance STUB1 ubiquitin ligase activity

    • Compounds that promote STUB1-RUNX1-RUNX1T1 interaction

    • Agents that selectively enhance degradation of leukemogenic fusion proteins

• Therapeutic monitoring applications:

  • Use STUB1 antibodies to monitor therapy response through:

    • Quantifying changes in STUB1-RUNX1-RUNX1T1 complexes during treatment

    • Measuring ubiquitination levels of RUNX1-RUNX1T1 as pharmacodynamic markers

    • Tracking subcellular redistribution of RUNX1-RUNX1T1 following treatment

• Potential combinatorial approaches:

  • Identify synergistic interactions between STUB1 activation and:

    • Proteasome inhibitors (timed administration to enhance STUB1-mediated ubiquitination before blocking degradation)

    • Hsp90 inhibitors (to release RUNX1-RUNX1T1 from chaperone protection)

    • Epigenetic modifiers affecting RUNX1-RUNX1T1 target genes

The development of STUB1-focused therapies presents a promising direction, particularly given that STUB1 expression is relatively low in RUNX1-RUNX1T1 leukemia cells compared to other leukemic cell lines , suggesting a potential therapeutic window for intervention.

What methodological advances are needed to fully characterize the role of STUB1 in cellular senescence pathways?

Current research has identified STUB1's involvement in cellular senescence through its interaction with BMAL1 , but several methodological advances would facilitate a more comprehensive understanding:

• Temporal analysis tools:

  • Develop real-time reporters of STUB1 activity during senescence progression

  • Create inducible systems to modulate STUB1 at specific stages of senescence

  • Establish single-cell analysis methods to account for heterogeneity in senescent populations

• Substrate identification approaches:

  • Apply global ubiquitinome analysis to identify the complete set of STUB1 substrates in senescent cells

  • Develop biotin proximity labeling methods to identify short-lived STUB1 interactions unique to senescent states

  • Create computational models integrating proteomic and transcriptomic data to predict senescence-specific STUB1 substrates

• Tissue-specific considerations:

  • Adapt methodologies for analyzing STUB1 function in difficult-to-culture cell types prone to senescence (e.g., neurons, cardiomyocytes)

  • Develop in situ techniques to study STUB1 activity in intact tissues

  • Create animal models with tissue-specific modulation of STUB1 activity

• Integration with senescence pathways:

  • Establish assays linking STUB1 activity to known senescence markers (p16, p21, SASP factors)

  • Develop methods to distinguish between STUB1's role in replicative, stress-induced, and oncogene-induced senescence

  • Create experimental systems to test the relationship between STUB1-mediated regulation of BMAL1/circadian rhythm and senescence timing

• Translational methodologies:

  • Develop techniques to assess STUB1 activity in patient-derived samples

  • Create screening platforms for compounds that modulate STUB1 activity in senescence contexts

  • Establish biomarkers for STUB1 pathway activation in aging-associated pathologies

The advancement of these methodologies would significantly enhance our understanding of how STUB1 contributes to cellular senescence and potentially reveal new therapeutic targets for age-related diseases.

What experimental designs would best elucidate the context-dependent functions of STUB1 across different tissue and cell types?

Understanding STUB1's context-dependent functions requires sophisticated experimental designs that account for tissue-specific factors:

• Comparative systems biology approach:

  • Perform parallel analyses of STUB1 interactomes across multiple cell types

  • Create tissue-specific STUB1 knockout/knockin animal models

  • Develop computational frameworks to predict tissue-specific STUB1 substrates based on proteomic data

• Microenvironment considerations:

  • Design co-culture systems to study STUB1 function at tissue interfaces

  • Develop 3D organoid models with STUB1 reporters

  • Create systems to modulate physical parameters (stiffness, oxygen tension) while monitoring STUB1 activity

• Developmental timing analyses:

  • Establish temporally controlled STUB1 modulation during differentiation

  • Create lineage-tracing tools linked to STUB1 activity

  • Develop methods to isolate stage-specific STUB1 complexes during development

• Disease model integration:

  • Compare STUB1 function across normal, pre-malignant, and malignant states

  • Develop patient-derived xenograft models with STUB1 modulation

  • Create synthetic lethality screens to identify context-dependent STUB1 vulnerabilities

• Multi-omics integration:

  • Combine ubiquitinome, proteome, transcriptome, and metabolome analyses

  • Develop computational methods to identify tissue-specific STUB1 regulatory networks

  • Create visualization tools for complex STUB1-dependent cellular processes

These experimental approaches would help resolve contradictory findings about STUB1 function across different biological contexts and potentially reveal new therapeutic opportunities for targeting STUB1 in a tissue-specific manner.