USP10 Antibody, HRP conjugated

What is USP10 and what are its main cellular functions?

USP10 (ubiquitin specific peptidase 10, also known as USP32 and NY-REN-60) is a member of the peptidase C19 family that functions as a deubiquitinating enzyme, removing ubiquitin from target proteins to prevent their degradation . The cellular functions of USP10 include:

• Regulation of p53 stability and localization through deubiquitination, particularly of cytoplasmic p53

• Protection of pancreatic cancer cells from endoplasmic reticulum stress, which may contribute to tumor progression

• Promotion of fibronectin recycling, secretion, and organization through deubiquitination of integrin subunits β1 and β5

• Involvement in paraptotic cell death pathways, potentially offering alternative therapeutic approaches for apoptosis-resistant cancers

• Deubiquitination of specific ribosomal protein subunits, including RPS2 and RPS3, affecting ribosomal recycling

Understanding these functions is essential when utilizing USP10 antibodies in research to properly interpret experimental results across different contexts.

What are the recommended dilutions for USP10 antibody, HRP conjugated, in various experimental applications?

Based on established protocols, the following dilutions are recommended for optimal results with USP10 antibody:

• Western Blot (WB): 1:1000-1:5000 dilution

• Immunofluorescence (IF)/Immunocytochemistry (ICC): 1:50-1:500 dilution

It's important to note that these ranges provide starting points for optimization. The ideal dilution may vary depending on:

• The specific cell line or tissue being analyzed

• The expression level of USP10 in your experimental system

• The detection method being used

• Sample preparation methods

A titration experiment is strongly recommended when first using the antibody in a new experimental system to determine the optimal concentration that provides the best signal-to-noise ratio .

What sample types have been validated for USP10 antibody detection?

The USP10 antibody has been validated for the following sample types:

The antibody shows reliable reactivity with both human and mouse samples . When working with tissues or cell lines not listed above, preliminary validation experiments are recommended to confirm specificity and optimal conditions.

What is the expected molecular weight of USP10 in Western blot applications?

When using the USP10 antibody in Western blot applications, researchers should be aware of the following molecular weight specifications:

• Calculated Molecular Weight: 87 kDa, 92 kDa

• Observed Molecular Weight: 100-130 kDa

The discrepancy between calculated and observed molecular weights is common for many proteins and can be attributed to:

• Post-translational modifications like phosphorylation, glycosylation, or ubiquitination

• The presence of isoforms or splice variants

• Incomplete denaturation during sample preparation

Researchers should expect a band between 100-130 kDa when performing Western blot analysis of USP10, and should be cautious about bands appearing at significantly different molecular weights unless validating specific isoforms.

How can I effectively use USP10 antibody to study its role in cancer progression?

USP10 has emerged as a significant factor in cancer biology, particularly in pancreatic ductal adenocarcinoma (PDAC) where its overexpression correlates with poor patient outcomes . When investigating USP10's role in cancer using antibody-based approaches, consider the following methodological strategies:

• Compare expression levels between normal and cancerous tissues:

  • Perform immunohistochemistry (IHC) on tissue microarrays with the standardized H-score method, which accounts for both staining intensity and percentage of positive cells

  • Verify IHC findings with Western blot analysis of tissue lysates

• Correlate USP10 expression with clinical outcomes:

  • Analyze The Cancer Genome Atlas (TCGA) data in conjunction with your antibody-based findings to establish clinical relevance

  • Stratify patients by USP10 expression levels and correlate with survival data

• Investigate mechanistic pathways:

  • Use the antibody in combination with USP10 knockdown studies to validate specificity

  • Examine downstream effects on ER stress markers (BiP, PERK, IRE1α) after USP10 silencing

  • Analyze ubiquitination status of known USP10 substrates like RPS3 and RPS2

• Perform rescue experiments:

  • Test whether chemical chaperones like TUDCA can reverse effects of USP10 silencing on ER stress markers

  • Use the antibody to confirm USP10 levels in rescue experiments

This multi-faceted approach allows for comprehensive assessment of USP10's contributions to cancer pathophysiology while maintaining experimental rigor.

What methodological approaches can help distinguish between nuclear and cytoplasmic USP10 functions?

USP10 exhibits differential localization patterns that correspond to distinct cellular functions. In unstressed cells, USP10 primarily localizes to the cytoplasm, while stress conditions can trigger nuclear translocation . To differentiate between nuclear and cytoplasmic USP10 functions:

• Subcellular fractionation followed by Western blotting:

  • Prepare separate nuclear and cytoplasmic fractions using standard protocols

  • Perform Western blot with USP10 antibody on each fraction

  • Include proper loading controls for each fraction (e.g., GAPDH for cytoplasm, Lamin B for nucleus)

• Immunofluorescence with co-localization analysis:

  • Utilize the USP10 antibody at 1:50-1:500 dilution for IF/ICC

  • Counter-stain with nuclear markers (DAPI/Hoechst)

  • Employ confocal microscopy for accurate subcellular localization

  • Quantify nuclear/cytoplasmic ratios under different experimental conditions

• Proximity ligation assay (PLA):

  • Use USP10 antibody in combination with antibodies against known nuclear (p53) or cytoplasmic (integrin) binding partners

  • PLA signals will reveal compartment-specific interactions

• Stress-response experiments:

  • Induce cellular stress (DNA damage, ER stress, etc.)

  • Monitor USP10 translocation using IF/ICC or fractionation approaches

  • Correlate localization changes with functional outcomes

These approaches enable researchers to dissect compartment-specific functions of USP10 and understand how its localization impacts its diverse cellular roles.

How can I effectively investigate USP10's interaction with p53 using antibody-based methods?

USP10 has been identified as a key regulator of p53, particularly in the context of the DNA damage response and tumor development . To investigate this important interaction:

• Co-immunoprecipitation (Co-IP):

  • Immunoprecipitate using either anti-USP10 or anti-p53 antibodies

  • Perform Western blot analysis on immunoprecipitates using the reciprocal antibody

  • Include appropriate controls including IgG control and USP10/p53-deficient cell lines (e.g., HCT116 p53-/- cells)

  • Consider performing IP under both basal and stress-induced conditions to capture dynamic interactions

• Proximity ligation assay (PLA):

  • Use USP10 and p53 antibodies to detect protein-protein interactions in situ

  • Quantify PLA signals under different experimental conditions

  • Compare cytoplasmic versus nuclear interaction signals

• Deubiquitination assays:

  • Immunoprecipitate p53 following treatment with proteasome inhibitors

  • Analyze ubiquitination status using anti-ubiquitin antibodies

  • Compare p53 ubiquitination with and without USP10 knockdown/overexpression

  • Use the USP10 antibody to confirm knockdown/overexpression efficiency

• Functional rescue experiments:

  • Silence endogenous USP10 and express mutant versions (e.g., catalytically inactive)

  • Use the antibody to validate expression levels of mutant proteins

  • Assess p53 stability, localization, and activity with established p53 assays

These methodological approaches provide complementary data on the USP10-p53 interaction and its functional consequences in various cellular contexts.

How can I troubleshoot inconsistent USP10 signal in Western blot experiments?

When encountering variable or weak USP10 signals in Western blot applications, consider addressing the following potential issues:

• Sample preparation:

  • Ensure complete cell lysis using RIPA or other appropriate buffers

  • Add protease inhibitors to prevent degradation

  • Include phosphatase inhibitors if phosphorylated forms are important

  • Maintain cold temperatures throughout preparation

• Protein loading and transfer:

  • Confirm equal protein loading using housekeeping proteins (GAPDH, β-actin)

  • Verify transfer efficiency with reversible membrane staining (Ponceau S)

  • Optimize transfer conditions for large proteins (USP10 is 100-130 kDa)

  • Consider using PVDF membranes instead of nitrocellulose for better protein retention

• Antibody conditions:

  • Titrate antibody concentration between 1:1000-1:5000

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

  • Test different blocking agents (5% milk vs. 5% BSA)

  • Ensure secondary antibody compatibility with HRP-conjugated primary

• Detection optimization:

  • Use enhanced chemiluminescence (ECL) substrates appropriate for your expected signal intensity

  • Adjust exposure times based on signal strength

  • Consider using signal enhancers for weak signals

If USP10 signal remains inconsistent after addressing these factors, validating antibody specificity using USP10 knockdown or knockout samples is recommended to ensure the observed bands are indeed USP10.

What are the optimal conditions for detecting USP10 in immunofluorescence studies?

For successful immunofluorescence detection of USP10:

• Cell fixation and permeabilization:

  • Test multiple fixation methods (4% paraformaldehyde, methanol, or acetone)

  • Optimize permeabilization conditions (0.1-0.5% Triton X-100 or 0.05-0.1% Saponin)

  • Fixation time and temperature can significantly impact epitope accessibility

• Antibody dilution and incubation:

  • Start with recommended 1:50-1:500 dilution range

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

  • Consider using amplification systems for weak signals

• Background reduction strategies:

  • Increase blocking time (1-2 hours with 5% normal serum)

  • Add 0.1-0.3% Triton X-100 to blocking and antibody dilution buffers

  • Include 0.05% Tween-20 in wash buffers

  • Perform additional wash steps to reduce non-specific binding

• Counterstaining considerations:

  • Use nuclear staining (DAPI/Hoechst) to provide context for USP10 localization

  • Consider co-staining with markers for specific cellular compartments (ER, mitochondria)

  • When examining stress responses, include markers for stress granules or DNA damage

For HepG2 cells specifically, which have been validated for USP10 detection , initial optimization should begin with manufacturer's recommended protocols before adjusting parameters as needed.

How can I validate the specificity of USP10 antibody signal in my experimental system?

• Genetic approaches:

  • Perform siRNA-mediated knockdown of USP10 (previously validated in studies)

  • Use CRISPR/Cas9 to generate USP10 knockout cell lines

  • Compare antibody signal between control and knockdown/knockout samples

  • Rescue experiments with exogenous USP10 expression

• Peptide competition:

  • Pre-incubate the antibody with immunizing peptide/protein

  • Compare signal between blocked and unblocked antibody

  • Specific signals should be significantly reduced with peptide competition

• Multiple antibodies approach:

  • Compare staining patterns using antibodies targeting different USP10 epitopes

  • Consistent patterns across antibodies support specificity

• Cross-reactivity assessment:

  • Test the antibody in tissues/cells known to lack USP10 expression

  • Include species controls if working across different organisms

  • The antibody has been validated for both human and mouse reactivity

• Correlation with mRNA expression:

  • Compare protein detection patterns with USP10 mRNA expression data

  • Concordance between protein and mRNA suggests specific detection

These validation approaches should be documented and included in publications to support the reliability of results obtained using the USP10 antibody.

How can I use USP10 antibody to investigate its role in regulating ribosomal protein deubiquitination?

USP10 has been shown to deubiquitinate specific ribosomal proteins, particularly RPS2 and RPS3, influencing ribosomal recycling . To investigate this function:

• Ubiquitination status analysis:

  • Immunoprecipitate specific ribosomal proteins (RPS2, RPS3, RPS10)

  • Probe for ubiquitin modification using anti-ubiquitin antibodies

  • Compare ubiquitination levels between control and USP10-manipulated samples

  • Use the USP10 antibody to confirm knockdown/overexpression efficiency

• Ribosomal fraction analysis:

  • Isolate ribosomal fractions using sucrose gradient centrifugation

  • Analyze USP10 association with ribosomes by Western blotting

  • Examine ribosomal protein ubiquitination in different fractions

• Protein synthesis assessment:

  • Implement the surface sensing of translation (SunSET) assay using puromycin incorporation

  • Detect puromycin-labeled peptides via anti-puromycin antibody Western blotting

  • Compare global protein synthesis rates in control versus USP10-manipulated cells

  • Follow established protocols using 10 μg/ml puromycin for 10 minutes prior to harvesting cells

• ER stress analysis:

  • Examine markers of ER stress (BiP, PERK, IRE1α) by Western blotting

  • Compare stress responses in control versus USP10-manipulated cells

  • Test whether chemical chaperones like TUDCA can rescue effects of USP10 silencing

These approaches will help elucidate USP10's role in maintaining ribosomal function and protein synthesis through deubiquitination of key ribosomal proteins.

What methods can I use to study USP10's involvement in paraptosis using the antibody?

Paraptosis is a form of programmed cell death characterized by cytoplasmic vacuolation and mitochondrial/ER dilation, offering potential therapeutic approaches for apoptosis-resistant cancers . To investigate USP10's role in paraptosis:

• Morphological assessment:

  • Utilize YFP-Mito cells which express fluorescence in mitochondria

  • Monitor mitochondrial dilation (a key paraptotic feature) under different conditions

  • Use the USP10 antibody in immunofluorescence to correlate USP10 levels with morphological changes

  • Combine with ER markers to examine ER dilation concurrently

• USP10 manipulation approaches:

  • Perform siRNA-mediated knockdown of USP10

  • Use spautin-1, a USP10 inhibitor, to pharmacologically inhibit USP10 activity

  • Monitor the effect of these interventions on paraptosis induction

  • Validate knockdown/inhibition efficiency using the USP10 antibody

• Stress induction experiments:

  • Treat cells with paraptosis inducers like curcumin

  • Compare paraptotic responses between control and USP10-manipulated cells

  • Use time-course experiments to determine critical windows for USP10 involvement

  • Employ the USP10 antibody to monitor expression levels throughout the process

• Rescue experiments:

  • Overexpress wild-type or mutant USP10 following knockdown

  • Determine which domains/activities of USP10 are essential for its role in paraptosis

  • Validate expression using the USP10 antibody in Western blot or immunofluorescence

These methodological approaches will help elucidate USP10's specific contributions to paraptotic cell death pathways and potentially identify new therapeutic targets.

How can I use USP10 antibody to study its role in integrin and matrix protein recycling?

USP10 plays an important role in promoting fibronectin (FN) recycling, secretion, and organization through its deubiquitinating activity on integrin subunits . To investigate this function:

• Integrin deubiquitination analysis:

  • Immunoprecipitate integrin subunits (β1, β5)

  • Detect ubiquitination status using anti-ubiquitin antibodies

  • Compare ubiquitination levels between control and USP10-manipulated samples

  • Use USP10 antibody to confirm knockdown/overexpression efficiency

• Integrin recycling assays:

  • Implement live cell confocal integrin recycling assays

  • Compare recycling rates between control and USP10-manipulated cells

  • Validate findings with cell surface biotinylation techniques

  • Quantify differences in integrin recycling (e.g., αvβ1β5) following USP10 manipulation

• Fibronectin trafficking studies:

  • Use biotinylated FN to track internalization and recycling

  • Measure internalization (2.1-fold increase with USP10 overexpression)

  • Analyze recycling over time (1.7–2.2-fold increase with USP10 overexpression)

  • Validate the role of integrins using blocking antibodies against α5β1 and αv integrins

• Extracellular matrix organization assessment:

  • Analyze FN organization through immunofluorescence

  • Quantify FN fiber formation and alignment

  • Determine the proportion of recycled versus newly secreted FN in the matrix

  • Correlate matrix organization with USP10 expression levels

These methodological approaches will provide comprehensive insights into USP10's role in regulating integrin trafficking and matrix protein organization, with implications for understanding tissue remodeling and fibrosis.

How can USP10 antibody be used to assess its potential as a biomarker in cancer tissues?

Given the correlation between USP10 overexpression and poor patient outcomes in certain cancers , investigating its potential as a biomarker is of clinical interest:

• Tissue microarray analysis:

  • Perform immunohistochemistry using standardized protocols

  • Implement the H-score system, which accounts for both staining intensity and percentage of positive cells

  • Compare expression levels across normal tissue, precancerous lesions, and cancer specimens

  • Correlate with clinical parameters (stage, grade, survival)

• Statistical validation approaches:

  • Use linear mixed models to assess differences in H-scores between groups

  • Perform Kaplan-Meier survival analysis stratifying patients by USP10 expression

  • Conduct multivariate analysis to determine independent prognostic value

  • Establish optimal cutoff values for high versus low expression

• Multi-marker panel development:

  • Combine USP10 IHC with other established or emerging biomarkers

  • Assess the added predictive value of USP10 in biomarker panels

  • Compare with standard clinical parameters

• Correlation with molecular profiling:

  • Compare USP10 protein expression with RNA-seq data from repositories like TCGA

  • Validate findings against multiple databases (GTEx, CCLE, ICGC, TCGA)

  • Identify patient subgroups with particular molecular features associated with USP10 overexpression

These rigorous approaches will help determine whether USP10 has value as a prognostic or predictive biomarker in specific cancer types, potentially informing clinical decision-making and treatment strategies.

What are the best practices for analyzing USP10 expression across different cancer types using the antibody?

When conducting comparative studies of USP10 expression across cancer types:

• Sample standardization:

  • Use tissue microarrays containing multiple cancer types and matched normal tissues

  • Implement consistent fixation and processing protocols

  • Include internal reference controls in each batch

  • Process all samples simultaneously when possible to minimize batch effects

• Quantification methods:

  • Employ digital pathology and automated image analysis for objective quantification

  • Use standardized scoring systems like H-score

  • Validate visual scoring with multiple independent observers

  • Calculate intra- and inter-observer variability

• Multiple detection methods:

  • Complement IHC with Western blot analysis when tissue lysates are available

  • Consider multiplex immunofluorescence to examine co-expression with other markers

  • Validate protein expression findings with publicly available transcriptomic data

• Comparative analysis framework:

  • Analyze expression across:

    • Normal vs. tumor tissue

    • Different cancer types (e.g., pancreatic vs. breast cancer)

    • Primary tumors vs. metastases

    • Different histological subtypes within the same cancer type

  • Correlate with molecular subtypes and driver mutations

This comprehensive approach enables reliable comparison of USP10 expression patterns across cancer types, potentially revealing cancer-specific roles and therapeutic implications.