usp109 Antibody

What is USP10 and what are the recommended applications for USP10 antibodies?

USP10 (Ubiquitin Specific Peptidase 10) belongs to the peptidase C19 family and functions as a deubiquitinase that removes ubiquitin from target proteins. USP10 antibodies are validated for multiple research applications:

It is recommended that researchers titrate these antibodies in each testing system to obtain optimal results as they may be sample-dependent .

How should researchers validate USP10 antibody specificity?

A comprehensive validation approach should include:

• Knockout/Knockdown Controls: Generate USP10 KO/KD cell lines using CRISPR/Cas9 or siRNA methods. For example, researchers successfully created USP10 KO A549 cell lines to validate antibody specificity, observing downregulation of Beclin-1 in these cells .

• Western Blot Analysis: USP10 typically shows bands at approximately 100-130 kDa (observed molecular weight), though the calculated molecular weight is 87-92 kDa. Verify antibody specificity by comparing wild-type and USP10-depleted samples .

• Cross-Reactivity Testing: Confirm species reactivity. Available data shows most USP10 antibodies react with human, mouse, and rat samples .

• Positive Controls: Include samples known to express USP10, such as MCF-7 or HEK-293 cells, which have been validated as positive controls for USP10 detection .

How can USP10 antibodies be used to study B cell immune responses and antibody diversification?

USP10 plays a critical role in B cell responses through its stabilization of activation-induced cytidine deaminase (AID). Research methodologies using USP10 antibodies include:

• Immunoblotting to Monitor AID Levels: USP10 antibodies can be used in knockout studies to demonstrate that USP10 deficiency significantly decreases AID protein levels, subsequently reducing neutralizing antibody production after immunization with SARS-CoV-2 or HIV-1 vaccines .

• Nuclear Translocation Analysis: Anti-USP10 antibodies can detect USP10's nuclear translocation following phosphorylation at T674 within its NLS domain. This translocation is synergistically promoted by signals from BCR and TLR1/2 receptors .

• Affinity Maturation Studies: In mouse models with USP10 knockout in B cells (USP10-B KO), antibodies can be used to track USP10 expression and correlate it with decreased IgG1 and IgA class-switching, impaired somatic hypermutation, and reduced antibody affinity maturation .

Experimental data shows:

What are the recommended protocols for studying USP10's role in deubiquitination processes?

For effective analysis of USP10's deubiquitination activity:

• Immunoprecipitation-Based Deubiquitination Assays:

  • Transfect cells with plasmids expressing the target protein along with HA-ubiquitin

  • Treat cells with proteasome inhibitors (e.g., MG-132) for 4-6 hours before harvest

  • Lyse cells in denaturing conditions (containing 1% SDS) with heat treatment

  • Dilute lysates and immunoprecipitate the target protein

  • Perform Western blot analysis with anti-HA antibodies to detect ubiquitination and anti-USP10 antibodies to confirm USP10 presence

• In Vitro Deubiquitination Assays:

  • Purify recombinant USP10 and ubiquitinated substrate proteins

  • Incubate USP10 with ubiquitinated substrates in deubiquitination buffer

  • Analyze reaction products by immunoblotting with specific antibodies

Research has shown that USP10 specifically stabilizes nuclear AID protein and affects Beclin-1 ubiquitination levels, demonstrating its importance in regulating protein stability through deubiquitination .

How can USP10 antibodies be utilized to investigate tumor immunology and cancer prognosis?

USP10 plays significant roles in cancer immunity and prognosis. Methods for investigating these functions include:

• Tumor Microenvironment Analysis:

  • Apply multiplex immunohistochemistry with USP10 antibodies alongside immune cell markers

  • Data shows USP10 expression correlates significantly with markers of dendritic cells, neutrophils, macrophages, CD4+ T cells, CD8+ T cells, and B cells in pancreatic adenocarcinoma (PAAD) and liver hepatocellular carcinoma (LIHC)

• Prognostic Correlation Studies:

  • Use USP10 antibodies in tissue microarrays of various cancer types

  • Research indicates high USP10 expression correlates with poor prognosis in PAAD, LIHC, LUAD, and BRCA

  • USP10 expression is also associated with cancer stage and lymph node metastasis

• Immune Cell Infiltration Assessment:

  • Correlation analysis shows USP10 expression is significantly associated with:

    • Dendritic cells (r = 0.4, P = 8.68e-11)

    • Neutrophils (r = 0.267, P = 4.64e-07)

    • Macrophages (r = 0.273, P = 2.47e-07)

    • CD4+ T cells (r = 0.118, P = 2.81e-02)

    • CD8+ T cells (r = 0.188, P = 4.46e-04)

    • B cells (r = 0.264, P = 6.78e-07) in LIHC

What are the recommended protocols for studying USP10's role in mucosal immunity following vaccination?

To investigate USP10's involvement in mucosal immunity after vaccination:

• Collection and Processing of Mucosal Samples:

  • Collect oral mucosal fluid samples at regular intervals (e.g., days 5, 10, 15, and 20) after vaccination

  • For nasal mucosal fluid collection, use specialized nasal swabs or lavage techniques

  • Process samples immediately or store at -80°C with protease inhibitors

• Detection of SARS-CoV-2 IgG Antibodies in Mucosal Samples:

  • Use enzyme-linked immunosorbent assays (ELISA) to quantify antibody levels

  • Research shows SARS-CoV-2 IgG antibodies can be detected in oral mucosal fluid with concentrations of approximately 153.4 ±141.0 ng/mL and in nasal mucosal fluid at 2496.0 ±2698.0 ng/mL following COVID-19 mRNA vaccination

• USP10 Regulation of Antibody Production:

  • Use USP10 antibodies to track expression in B cells from mucosal tissues

  • Combined with knockout studies, researchers can correlate USP10 levels with antibody production

  • The deficiency of USP10 in B cells significantly decreases neutralizing antibody production after immunization with SARS-CoV-2 or HIV-1 nanoparticle vaccines

What techniques are recommended for studying USP10 in Schizosaccharomyces pombe models?

For researchers using S. pombe as a model system:

• Antibody Pull-Down Experiments:

  • Follow established protocols for detecting protein-protein interactions in fission yeast

  • Use appropriate lysis buffers containing protease inhibitors to preserve protein interactions

  • Perform immunoprecipitation with anti-USP10 antibodies followed by mass spectrometry to identify interacting partners

• Gene Targeting and Manipulation:

  • Use CRISPR/Cas9 technology for efficient gene targeting

  • The efficiency of gene targeting can be increased to 75-80% by removing pku70+ and pku80+ genes

  • For expression studies, vectors like pINTL, pINTK, and pINTH can be used for targeted integration

• RNA Isolation and Expression Analysis:

  • Extract total RNA using High Pure RNA Isolation kit

  • Perform quantitative RT-PCR using specific primers

  • Use reference genes such as act1 for normalization

What are common issues when using USP10 antibodies and how can they be addressed?

How can researchers study USP10's involvement in nuclear events and protein regulation?

USP10 translocation to the nucleus is crucial for its function in regulating nuclear proteins like AID. Recommended approaches include:

• Nuclear/Cytoplasmic Fractionation:

  • Prepare separate nuclear and cytoplasmic extracts

  • Analyze USP10 distribution by Western blot

  • Include controls such as lamin B (nuclear marker) and tubulin (cytoplasmic marker)

  • Research shows anti-μ antibody treatment slightly increases USP10 nuclear translocation, but co-treatment with anti-μ antibody and TLR ligands has a synergistic effect

• Phosphorylation Analysis:

  • USP10 nuclear translocation depends on AKT-mediated phosphorylation at T674 within its NLS domain

  • Use phospho-specific antibodies or phosphatase treatments to confirm the role of phosphorylation

  • Employ kinase inhibitors to block translocation

• Immunofluorescence Microscopy:

  • Use confocal microscopy with nuclear staining (DAPI) and anti-USP10 antibodies

  • Track USP10 localization under different stimulation conditions

  • Quantify nuclear/cytoplasmic ratios using appropriate imaging software

This comprehensive understanding of USP10's nuclear dynamics is essential for studying its role in regulating proteins involved in antibody diversification and immune responses .

USP10 Antibody Research and Applications in Immunology and Cancer

USP10 antibodies serve as crucial tools for investigating the roles of this deubiquitinase in diverse biological processes, including immune responses, cancer progression, and cellular regulation. This FAQ collection provides evidence-based methodological guidance for researchers at various levels of expertise.