USP10 Antibody, FITC conjugated

What is USP10 and what are its primary functions in cellular pathways?

USP10 belongs to the peptidase C19 family and functions as a deubiquitinating protease. Its primary biological roles include:

• Regulation of p53 in DNA damage response and tumor development, where it deubiquitinates p53, reversing Mdm2-induced p53 nuclear export and degradation

• Stabilization of CD36 protein through deubiquitination, promoting lipid uptake and foam cell formation in macrophages, which is relevant to atherosclerosis progression

• After DNA damage, USP10 becomes stabilized and partially translocates to the nucleus to activate p53

These multiple roles make USP10 an important target for research in cancer biology, cardiovascular disease, and cellular stress response pathways.

What are the typical applications for USP10 antibodies in research?

Based on published literature and manufacturer data, USP10 antibodies are validated for multiple applications:

For FITC-conjugated USP10 antibodies specifically, flow cytometry and fluorescence microscopy would be the primary applications, leveraging the green fluorescence properties of FITC.

What species reactivity can I expect with USP10 antibodies?

Most commercially available USP10 antibodies demonstrate reactivity with:

• Human samples

• Mouse samples

• Rat samples

This cross-reactivity has been confirmed through both manufacturer testing and citation in published research . When designing experiments with animal models, this broad reactivity allows for translational research between human and rodent systems.

How does USP10 interaction with CD36 influence atherosclerosis development, and how can I study this with fluorescently labeled antibodies?

USP10 plays a critical role in atherosclerosis progression through its interaction with CD36, a scavenger receptor responsible for approximately 50% of lipid uptake in monocytes. Research findings indicate:

• USP10 directly interacts with CD36 as demonstrated through co-immunoprecipitation (Co-IP) experiments

• USP10 stabilizes CD36 protein by cleaving its poly-ubiquitin chains, preventing CD36 degradation

• Inhibition or knockdown of USP10 reduces CD36 protein levels without affecting its mRNA expression, suggesting post-translational regulation

• Confocal microscopy confirms co-localization of USP10 and CD36

To study this interaction using FITC-conjugated USP10 antibodies:

• Perform double immunofluorescence staining with FITC-conjugated USP10 antibody and a different fluorophore-conjugated CD36 antibody

• Use confocal microscopy to analyze co-localization patterns

• Combine with proximity ligation assays (PLA) to confirm direct protein-protein interactions

• Implement flow cytometry with FITC-conjugated USP10 antibody to quantify expression levels after treatments with lipids or USP10 inhibitors

This approach allows visualization of both proteins' expression patterns and their potential interaction sites within macrophages or vascular cells.

What are the technical considerations when using USP10 antibody in deubiquitination assays, and how does the FITC conjugation impact experimental design?

When studying USP10's deubiquitinating activity, several technical factors must be considered:

• Substrate specificity: USP10 cleaves ubiquitin chains from specific substrate proteins. Fluorogenic substrates like Ub-AMC are useful for studying general DUB activity, where AMC fluorescence increases upon deubiquitination

• Activity preservation:

  • The catalytic site of USP10 must remain accessible and functional

  • Buffer conditions should maintain enzymatic activity (typically PBS with specific additives)

  • FITC conjugation should be designed to avoid the catalytic domain

• Dual fluorophore considerations with FITC-conjugated antibodies:

  • When using FITC-conjugated USP10 antibody in conjunction with AMC-based substrates, spectral overlap must be addressed

  • FITC: λex/λem ≈ 495/519 nm

  • AMC: λex/λem ≈ 350/460 nm

  • Design experiments with appropriate filter sets to discriminate between signals

• Functional validation approach:

  • Pre-incubate USP10 with or without test inhibitors

  • Add fluorogenic substrate (e.g., Ub-AMC)

  • Monitor fluorescence increase over time at appropriate wavelengths

  • Include controls with known inhibitors such as Ub-Aldehyde

For FITC-conjugated antibodies specifically, they are more suitable for localizing USP10 rather than directly studying enzymatic activity, as antibody binding might interfere with catalytic function.

How can I design experiments to investigate USP10's role in p53 regulation using fluorescence-based approaches?

USP10 has been identified as a critical regulator of p53, particularly following DNA damage. A comprehensive experimental design would include:

• Subcellular localization studies:

  • Track USP10 translocation to the nucleus after DNA damage using FITC-conjugated USP10 antibody

  • Co-stain with p53 antibodies (different fluorophore) to visualize potential co-localization

  • Implement time-course imaging after DNA damage induction with agents like etoposide or UV radiation

• Protein stability analysis:

  • Perform cycloheximide chase experiments to measure p53 half-life

  • Compare control cells with USP10-overexpressing or USP10-knockdown cells

  • Use FITC-conjugated USP10 antibody to confirm expression levels by flow cytometry

• Deubiquitination assay:

  • Immunoprecipitate p53 from cells

  • Probe for ubiquitin chains before and after DNA damage

  • Compare ubiquitination patterns in USP10-depleted versus control cells

• Live-cell imaging approach:

  • Express fluorescently-tagged USP10 and p53 proteins

  • Perform FRET analysis to detect direct interaction

  • Track protein dynamics in real-time after DNA damage

• Functional outcome measurement:

  • Assess p53 target gene expression using RT-qPCR

  • Evaluate cell cycle arrest and apoptosis in relation to USP10 levels

  • Correlate findings with USP10 subcellular distribution as determined by fluorescence microscopy

These methodologies can help establish the temporal and spatial relationships between USP10 and p53, providing mechanistic insights into this regulatory pathway.

What are the optimal fixation and permeabilization protocols for USP10 immunofluorescence staining with FITC-conjugated antibodies?

For optimal results with USP10 immunofluorescence using FITC-conjugated antibodies, consider these methodological recommendations:

• Fixation options:

  • 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature preserves both morphology and fluorescence

  • Methanol fixation (-20°C for 10 minutes) may better expose some epitopes but can reduce FITC fluorescence

  • For double staining of USP10 and CD36, PFA fixation is preferred as it preserves membrane proteins like CD36

• Permeabilization approaches:

  • For cytoplasmic USP10: 0.1-0.2% Triton X-100 for 5-10 minutes

  • For nuclear USP10 (post-DNA damage): 0.5% Triton X-100 may be required for better nuclear penetration

  • Alternative: 0.05% saponin for milder permeabilization that better preserves membranous structures

• Blocking conditions:

  • 5% normal serum (match to secondary antibody host if using indirect detection)

  • 1-3% BSA in PBS with 0.1% Tween-20

  • Include 0.3M glycine to reduce background, especially important for FITC which can have higher background

• Antibody dilutions and incubation:

  • For direct FITC-conjugated USP10 antibodies: 1:50-1:500 dilution range

  • Incubate overnight at 4°C in humid chamber protected from light

  • Include proper controls: isotype control and unstained samples

• Counterstaining considerations:

  • DAPI (blue) for nuclear visualization works well with FITC (green)

  • Avoid propidium iodide (red) which may have spectral overlap with FITC

Following these protocols should yield consistent and specific staining patterns for USP10 in various cell types, including HEK-293 cells which have been validated for positive immunofluorescence detection .

How can I validate USP10 antibody specificity for my research applications?

Thorough validation of USP10 antibody specificity is essential for generating reliable research data. A comprehensive validation strategy should include:

• Genetic approaches:

  • USP10 knockdown verification: Compare antibody signal in control vs. siRNA/shRNA USP10-depleted samples

  • USP10 knockout validation: Use CRISPR/Cas9-generated USP10 knockout cells as negative controls

  • Overexpression confirmation: Test detection of exogenously expressed USP10 (tagged versions can provide dual validation)

• Biochemical validations:

  • Western blot analysis: Confirm single band at expected molecular weight (100-130 kDa observed, though calculated weight is 87-92 kDa)

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide before application

  • Mass spectrometry validation: Identify USP10 in immunoprecipitated samples

• FITC-conjugated antibody-specific controls:

  • Fluorescence minus one (FMO) controls for flow cytometry

  • Photobleaching controls to distinguish true signal from autofluorescence

  • Isotype-matched FITC-conjugated control antibodies

• Cross-reactivity assessment:

  • Test reactivity in multiple species if working with animal models

  • Published literature confirms USP10 antibody reactivity with human, mouse, and rat samples

• Application-specific validations:

  • For IP: Confirm pulldown efficiency by Western blot

  • For IF: Compare staining patterns with multiple USP10 antibodies recognizing different epitopes

  • For functional studies: Correlate antibody detection with functional readouts (e.g., deubiquitinating activity)

Implementation of these validation steps ensures that experimental findings accurately reflect USP10 biology rather than potential antibody artifacts.

What are the optimal protocols for detecting USP10 in flow cytometry using FITC-conjugated antibodies?

Flow cytometry with FITC-conjugated USP10 antibodies requires careful protocol optimization for sensitive and specific detection. Here's a comprehensive approach:

• Cell preparation considerations:

  • Single cell suspension: Gentle enzymatic dissociation for adherent cells

  • Viability staining: Include far-red viability dye compatible with FITC channel

  • Fixation options: 2-4% paraformaldehyde for 10-15 minutes at room temperature

• Permeabilization strategy (for intracellular USP10):

  • Methanol-based: 90% ice-cold methanol for 30 minutes (better for nuclear proteins)

  • Detergent-based: 0.1% Triton X-100 or commercial permeabilization buffers

  • Saponin-based (0.1%): For reversible permeabilization with lower background

• Staining protocol optimization:

  • Antibody titration: Test dilution series from 1:50 to 1:500 to determine optimal signal-to-noise ratio

  • Incubation conditions: 30-60 minutes at room temperature or 4°C, protected from light

  • Washing steps: Minimum 2-3 washes with excess buffer to reduce background

• Instrument setup and controls:

  • FITC detection: 488nm laser excitation with 530/30nm bandpass filter

  • Required controls:

    • Unstained cells for autofluorescence assessment

    • Isotype-FITC control to establish background

    • FMO (Fluorescence Minus One) controls for multicolor panels

    • Positive control: Cell line with known high USP10 expression (e.g., MCF-7 or HEK-293 )

• Analysis considerations:

  • Gating strategy: Exclude debris, doublets, and dead cells before analyzing USP10 signal

  • Histogram visualization: Compare median fluorescence intensity (MFI) between experimental groups

  • Statistical analysis: Calculate fold change in MFI relative to control samples

This methodological approach allows for quantitative assessment of USP10 expression levels across different cell populations or experimental conditions, enabling correlations with functional outcomes in your research.

I'm experiencing high background when using FITC-conjugated USP10 antibody in immunofluorescence - what are the most effective troubleshooting steps?

High background is a common challenge with FITC-conjugated antibodies due to the fluorophore's brightness and susceptibility to photobleaching. Follow these systematic troubleshooting steps:

• Fixation and permeabilization optimization:

  • Reduce fixation time to minimize autofluorescence (10-15 minutes in 4% PFA)

  • Test milder permeabilization (0.1% vs 0.5% Triton X-100)

  • Consider saponin (0.05%) for gentler permeabilization with less background

• Blocking enhancement strategies:

  • Increase blocking time (1-2 hours at room temperature)

  • Use combination blocks (5% serum + 1% BSA)

  • Add 0.1-0.3M glycine to quench aldehyde groups from fixation

  • Include 0.05% Tween-20 in all buffers to reduce non-specific binding

• Antibody optimization:

  • Further dilute antibody (test range from 1:500 to 1:2000)

  • Reduce incubation time or temperature

  • Pre-adsorb antibody with cell/tissue powder from negative control samples

  • Try different FITC-conjugated USP10 antibody clones if available

• Washing protocol enhancements:

  • Increase wash volume and duration (4-5 washes, 5-10 minutes each)

  • Add 0.05-0.1% Tween-20 to wash buffers

  • Include one high-salt (500mM NaCl) wash to disrupt low-affinity binding

• Imaging adjustments:

  • Use narrower bandpass filters to reduce autofluorescence collection

  • Implement spectral unmixing if your microscope system supports it

  • Utilize structured illumination or confocal microscopy to reduce out-of-focus signal

  • Apply post-acquisition background subtraction algorithms

• Sample-specific considerations:

  • For tissues: Treat with Sudan Black B (0.1-0.3%) to reduce lipofuscin autofluorescence

  • For fixed cells: Include photobleaching step before antibody incubation

  • For specific cell types (e.g., macrophages): Block Fc receptors with appropriate blocking reagents

Implementation of these approaches, particularly focusing on the blocking and washing steps, should significantly improve signal-to-noise ratio for FITC-conjugated USP10 antibody staining.

How can I optimize western blot protocols when working with USP10 antibodies to detect the correct molecular weight band?

USP10 protein detection by western blot can be challenging due to the discrepancy between calculated (87-92 kDa) and observed (100-130 kDa) molecular weights . Follow these optimization strategies:

• Sample preparation refinements:

  • Use multiple lysis buffers to compare extraction efficiency (RIPA vs. NP-40 vs. Triton-based)

  • Include complete protease inhibitor cocktail plus specific DUB inhibitors (e.g., N-ethylmaleimide)

  • Maintain cold temperature throughout preparation to prevent degradation

  • Sonicate briefly to shear DNA and reduce sample viscosity

• Gel electrophoresis parameters:

  • Select appropriate gel percentage (8% acrylamide gels work well for 100-130 kDa proteins)

  • Extend running time to improve separation in the 100-130 kDa range

  • Include molecular weight markers that span 70-150 kDa range

  • Load positive control lysates from validated cell lines (MCF-7, HEK-293 )

• Transfer optimization:

  • For large proteins like USP10, use wet transfer systems rather than semi-dry

  • Reduce methanol concentration in transfer buffer to 10% for better transfer of large proteins

  • Extend transfer time (overnight at low voltage/4°C can improve results)

  • Consider using PVDF membrane (0.45 μm pore size) instead of nitrocellulose for better protein retention

• Blocking and antibody incubation:

  • Test both BSA and milk-based blocking solutions (milk may contain phosphatases that affect modified proteins)

  • Optimize primary antibody dilution (1:500-1:1000 for polyclonal or 1:5000-1:50000 for monoclonal )

  • Extend primary antibody incubation (overnight at 4°C)

  • Include 0.05% Tween-20 in all buffers to reduce background

• Detection system considerations:

  • Use high-sensitivity ECL substrates for challenging targets

  • Consider fluorescent secondary antibodies for more quantitative analysis

  • Optimize exposure times to capture bands in the linear range

• Validation approaches:

  • Run USP10 knockdown/knockout samples in parallel to confirm specificity

  • If multiple bands appear, perform immunoprecipitation followed by western blot

  • Consider phosphatase treatment of lysates if post-translational modifications affect migration

Following these detailed protocol optimizations should help obtain clear and specific detection of USP10 at its characteristic 100-130 kDa observed molecular weight.

What approaches can I use to quantitatively measure USP10 deubiquitinating activity in experimental samples?

Measuring USP10's enzymatic activity requires specialized assays that can detect deubiquitination events. Here are comprehensive approaches for quantitative assessment:

• Fluorogenic substrate assays:

  • Substrate selection: Ub-AMC is a widely used fluorogenic substrate where AMC fluorescence increases upon deubiquitination

  • Reaction setup:

    • Purified USP10 or immunoprecipitated USP10 from experimental samples

    • Ub-AMC substrate at optimized concentration

    • Reaction buffer maintaining enzyme activity

  • Detection: Monitor fluorescence increase (λex/λem: 350/460 nm) over time

  • Quantification: Calculate reaction velocity from the slope of the fluorescence curve

• Cell-based deubiquitination assays:

  • Substrate-specific approach:

    • Immunoprecipitate specific USP10 substrates (e.g., p53 or CD36)

    • Perform western blot analysis with anti-ubiquitin antibodies

    • Compare ubiquitination levels across conditions

  • Reporter-based systems:

    • Utilize Ub-modified fluorescent proteins that change localization or fluorescence properties upon deubiquitination

    • Measure by flow cytometry or fluorescence microscopy

• Di-ubiquitin cleavage assays:

  • Use differentially labeled di-ubiquitin chains (K48, K63, etc.)

  • Incubate with USP10 (purified or immunoprecipitated)

  • Analyze chain cleavage by western blot or specialized FRET-based detection

  • Quantify chain-type specificity of USP10 activity

• Mass spectrometry-based approaches:

  • Global approach: Compare ubiquitinome profiles in USP10 wild-type versus knockout/knockdown cells

  • Targeted approach: Focus on specific substrates and quantify ubiquitin chain modifications

  • AQUA peptide strategy for absolute quantification of ubiquitin chain types

• In-cell activity probes:

  • Activity-based probes: Use Ub-based reactive probes that covalently attach to active DUBs

  • Cell penetrant probes allow assessment of USP10 activity in intact cells

  • Detection by gel electrophoresis or imaging techniques

• Inhibitor-based validation:

  • Include control reactions with known USP10 inhibitors or pan-DUB inhibitors (e.g., Ub-aldehyde)

  • Generate dose-response curves to quantify inhibition potency

  • Use as positive controls in activity assays

These methodologies provide complementary approaches to quantitatively assess USP10 activity, from purified enzyme systems to complex cellular environments, allowing correlation of enzymatic function with biological outcomes in your experimental system.

How can I design experiments to study the role of USP10 in atherosclerosis using FITC-conjugated antibodies and flow cytometry?

Based on USP10's role in CD36 stabilization and atherosclerosis progression , here's a comprehensive experimental design using FITC-conjugated USP10 antibodies:

• Cellular models setup:

  • Primary cell isolation: Monocytes from human blood or bone marrow-derived macrophages from mice

  • Differentiation protocol: Treat monocytes with M-CSF for macrophage differentiation

  • Foam cell induction: Expose macrophages to oxLDL (oxidized low-density lipoprotein)

• Experimental groups design:

  • Control macrophages (no treatment)

  • oxLDL-treated macrophages (foam cell induction)

  • USP10 inhibitor-treated + oxLDL

  • USP10 knockdown/knockout cells + oxLDL

  • USP10 overexpression + oxLDL

• Flow cytometry analysis protocol:

  • Multicolor panel design:

    • FITC-conjugated USP10 antibody

    • PE-conjugated CD36 antibody

    • Far-red lipid stain (e.g., LipidTOX Deep Red)

    • Viability dye compatible with other fluorophores

  • Gating strategy:

    • Exclude debris and doublets

    • Gate on viable cells

    • Analyze USP10 vs. CD36 expression

    • Correlate with lipid content

• Functional assessments:

  • Lipid uptake kinetics: Time-course analysis of fluorescently-labeled oxLDL uptake

  • Efflux capacity: Measure cholesterol efflux to apoA-I or HDL

  • Inflammatory profile: Quantify cytokine production in response to various treatments

  • Cell migration assays: Assess impact on macrophage chemotaxis

• Molecular mechanism investigation:

  • Co-immunoprecipitation: Confirm USP10-CD36 interaction under different conditions

  • Ubiquitination analysis: Measure CD36 ubiquitination status after USP10 modulation

  • Half-life determination: Conduct cycloheximide chase experiments to assess CD36 stability

• Translational approaches:

  • Human atherosclerotic plaque analysis: Immunostain for USP10 and CD36

  • Mouse models: Compare ApoE-/- mice with ApoE-/-/USP10-/- double knockouts

  • Ex vivo analysis: Flow cytometry of plaque-derived cells for USP10 and CD36 expression

This experimental design allows for comprehensive assessment of USP10's role in atherosclerosis progression, from molecular mechanisms to cellular phenotypes and potential therapeutic implications of USP10 inhibition.

What are the most effective experimental controls when using FITC-conjugated USP10 antibodies in confocal microscopy studies?

For rigorous confocal microscopy studies with FITC-conjugated USP10 antibodies, implement these essential controls:

• Specificity controls:

  • Genetic knockout/knockdown: USP10 siRNA-treated or CRISPR/Cas9 knockout cells

  • Peptide competition: Pre-incubation of antibody with immunizing peptide/protein

  • Multiple antibody validation: Compare staining pattern with different USP10 antibodies

  • Overexpression control: Cells transfected with tagged USP10 constructs

• Technical fluorescence controls:

  • Unstained samples: To establish autofluorescence baseline

  • Isotype control: FITC-conjugated isotype-matched irrelevant antibody

  • Single-color controls: For spectral unmixing in multicolor experiments

  • Signal specificity test: Secondary-only control (if using indirect detection)

• Biological condition controls:

  • Known expression pattern references: Include cell types with established USP10 expression (e.g., HEK-293, MCF-7)

  • Subcellular localization validation: Compare cytoplasmic vs. nuclear distribution before/after DNA damage

  • Stimulus response: Positive control for USP10 translocation (e.g., DNA damage induction)

• Imaging parameter controls:

  • Photobleaching assessment: Repeated imaging of the same field to quantify signal decay

  • Laser power series: Images at multiple excitation intensities to confirm linear response

  • Z-stack validation: Multiple focal planes to confirm true signal vs. out-of-focus artifacts

  • Channel bleed-through check: Sequential vs. simultaneous acquisition comparison

• Quantification controls:

  • Intensity calibration: Fluorescent beads with known intensity values

  • Background subtraction validation: Multiple methods comparison

  • Colocalization controls: Randomized image analysis to establish statistical thresholds

• Specialized controls for co-localization studies:

  • Positive co-localization control: Known interacting proteins

  • Negative co-localization control: Proteins in distinct subcellular compartments

  • Mathematical validation: Pearson's and Mander's coefficients compared to randomized datasets

How can I design experiments to investigate USP10's role in DNA damage response pathways using fluorescence-based approaches?

To investigate USP10's involvement in DNA damage response, particularly its regulation of p53 , design experiments using these fluorescence-based approaches:

• DNA damage induction and temporal dynamics:

  • Treatment options:

    • UV irradiation (10-50 J/m²)

    • Ionizing radiation (2-10 Gy)

    • Chemical agents: etoposide (10-50 μM), doxorubicin (0.2-1 μM), or cisplatin (5-20 μM)

  • Time-course design:

    • Short intervals for early events (15, 30, 60 minutes)

    • Extended timepoints for later responses (2, 4, 8, 24 hours)

• Subcellular translocation analysis:

  • Confocal microscopy with FITC-conjugated USP10 antibody to track:

    • Baseline cytoplasmic localization

    • Nuclear translocation after DNA damage

    • Co-localization with p53 (using different fluorophore)

  • Quantification approach:

    • Nuclear/cytoplasmic intensity ratio measurement

    • Time-dependent changes in compartmentalization

• Protein stability assessment:

  • Dual-color flow cytometry:

    • FITC-conjugated USP10 antibody

    • Alternative fluorophore-conjugated p53 antibody

  • Cycloheximide chase experiments:

    • Compare p53 half-life in USP10-depleted vs. control cells

    • Analyze by flow cytometry or western blotting at multiple timepoints

• Functional fluorescent reporters:

  • p53 transcriptional activity:

    • p53-responsive GFP reporter constructs

    • Measure activity in USP10 wild-type vs. knockout backgrounds

  • DNA damage sensors:

    • γH2AX foci quantification alongside USP10 staining

    • Live-cell imaging with fluorescent 53BP1 constructs

• Protein interaction visualization:

  • Proximity ligation assay (PLA):

    • Detect USP10-p53 interactions at single-molecule resolution

    • Quantify interaction frequency before/after DNA damage

  • FRET/FLIM approaches:

    • Express fluorescently-tagged USP10 and p53

    • Measure energy transfer as indicator of direct interaction

    • Track dynamic changes in real-time after damage

• Deubiquitination activity visualization:

  • Ubiquitin sensors:

    • Fluorescent ubiquitin chains as substrates

    • Monitor deubiquitination kinetics in cell extracts

  • In situ approaches:

    • Implement ubiquitin-specific fluorescent probes

    • Compare activity in nuclear vs. cytoplasmic compartments

• Cell fate correlation:

  • Multiplex analysis combining:

    • USP10 levels (FITC-conjugated antibody)

    • DNA damage markers (e.g., γH2AX)

    • Apoptosis indicators (Annexin V, caspase activity)

    • Cell cycle distribution (PI staining)

This comprehensive experimental approach enables detailed characterization of USP10's role in DNA damage response, connecting molecular mechanisms to cellular outcomes using quantitative fluorescence-based methodologies.

What are the validated dilution ranges and experimental conditions for using USP10 antibodies across different applications?

Based on manufacturer data and published research, here are the validated dilution ranges and experimental conditions for USP10 antibodies:

For FITC-conjugated versions, which are typically used for flow cytometry and IF/ICC, slight modifications to these ranges may be necessary due to the fluorophore's properties:

• Flow cytometry: Start with 1:100 dilution and titrate to optimal signal-to-noise ratio

• IF/ICC: Begin with 1:100 dilution, which typically requires less antibody than unconjugated versions

Experimental Conditions for Optimal Results:

• Western Blot Conditions:

  • Lysis buffer: RIPA buffer with protease inhibitors

  • Gel percentage: 8-10% acrylamide

  • Protein loading: 20-50 μg total protein

  • Observed molecular weight: 100-130 kDa

• Immunoprecipitation Conditions:

  • Lysis buffer: NP-40 or Triton X-100 based

  • Antibody binding: Overnight at 4°C

  • Protein A/G beads: 2-4 hours incubation

  • Washing buffer: TBS with 0.1% Tween-20

• Immunofluorescence/ICC Conditions:

  • Fixation: 4% paraformaldehyde, 10-15 minutes

  • Permeabilization: 0.1-0.2% Triton X-100, 5-10 minutes

  • Blocking: 5% normal serum, 1 hour

  • Primary antibody: Overnight at 4°C

  • Counterstain: DAPI for nuclear visualization

• IHC Conditions:

  • Antigen retrieval: TE buffer pH 9.0 (recommended) or citrate buffer pH 6.0

  • Detection system: HRP/DAB

  • Counterstain: Hematoxylin

These validated conditions provide a starting point for experimental design, though optimization may be necessary for specific experimental systems or when using FITC-conjugated variants of these antibodies.

What are the molecular interactions of USP10 that have been confirmed through co-immunoprecipitation studies?

USP10 has been demonstrated to interact with several key proteins through co-immunoprecipitation (Co-IP) studies, revealing its diverse functional roles:

• CD36 interaction:

  • Bidirectional Co-IP confirmed: USP10 antibody pulled down CD36 and CD36 antibody pulled down USP10

  • Functional significance: USP10 stabilizes CD36 by deubiquitination, promoting foam cell formation and atherosclerosis progression

  • Confirmed by confocal microscopy showing co-localization of FLAG-tagged USP10 with endogenous CD36

  • Mechanistic insight: USP10 inhibition or knockdown increased ubiquitinated CD36, accelerating its degradation

• p53 pathway interactions:

  • Direct binding to p53 demonstrated by Co-IP

  • Functional consequence: USP10 deubiquitinates p53, reversing Mdm2-induced nuclear export and degradation

  • DNA damage response: USP10 stabilization and partial nuclear translocation activates p53

  • Tumor suppressor role: USP10 may function as a regulator of p53 in cancer development

• Other validated interactions (from published literature):

  • CFTR (Cystic Fibrosis Transmembrane conductance Regulator): USP10 deubiquitinates and stabilizes CFTR

  • BECN1 (Beclin 1): USP10 regulates autophagy through deubiquitination of Beclin 1

  • SIRT6 (Sirtuin 6): Forms a complex regulating DNA damage response

  • SNX3 (Sorting Nexin 3): Involved in endosomal trafficking

The table below summarizes key interactions and their functional implications:

These validated interactions highlight USP10's multifaceted roles in cellular processes including lipid metabolism, DNA damage response, protein trafficking, and autophagy, making it an important target for research across multiple disease contexts.