USP15 Antibody

What are the validated applications for USP15 antibodies?

USP15 antibodies have been validated for multiple applications including Western Blot (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and Co-Immunoprecipitation (CoIP). Data shows that antibodies like 14354-1-AP have been tested in various cell lines including HEK-293, HeLa, HepG2, K-562, MCF-7, NIH/3T3 cells, as well as mouse and rat tissue samples . The specific applications and optimal dilutions vary by antibody:

It's important to note that optimal dilutions are sample-dependent and should be determined experimentally for each new system .

How do I choose between different USP15 antibody epitope targets?

When selecting a USP15 antibody, consider the specific domain you wish to target based on your research question:

• N-terminal targeting antibodies: These recognize the DUSP domain and are useful for detecting total USP15 protein regardless of modification state. They're particularly valuable when studying USP15's catalytic activity .

• C-terminal targeting antibodies: These recognize the C-terminal region (residues 740-981) which is important for protein-protein interactions, including the BARD1 interaction region. These antibodies are recommended when investigating USP15's role in DNA damage response pathways .

Make your selection based on whether you need to detect total USP15 or are interested in a specific functional domain. Cross-validate with multiple antibodies when critical findings depend on antibody specificity.

What species reactivity should I expect from USP15 antibodies?

Most commercially available USP15 antibodies demonstrate reactivity against human, mouse, and rat USP15 proteins. Some antibodies show broader cross-reactivity including bovine, dog, horse, rabbit, pig, and other species . For example:

• Antibody 14354-1-AP has confirmed reactivity with human, mouse, and rat samples, with cited applications in bovine systems as well .

• The ABIN6258347 antibody (N-Term) shows predicted reactivity with pig, bovine, horse, sheep, rabbit, dog, and chicken samples in addition to confirmed human, mouse, and rat reactivity .

Always verify species cross-reactivity when working with non-standard model organisms, as sequence conservation may vary across different regions of the protein.

How should I design experiments to study USP15's role in DNA damage repair?

To investigate USP15's function in DNA damage repair, consider the following experimental approach based on published methodologies:

• Generate USP15-deficient cells: Create CRISPR knockout (KO) or knockdown (KD) cells alongside appropriate controls (including rescue experiments with WT USP15 to confirm specificity) .

• Assess DNA damage sensitivity: Treat USP15-KO cells with various DNA-damaging agents (camptothecin, mitomycin C, hydroxyurea, or ionizing radiation) and measure cell survival using colony formation assays. USP15-deficient cells typically show hypersensitivity to these agents .

• Evaluate DNA damage markers: Monitor γH2AX foci formation by immunofluorescence at early (0-1h) and late (24h) timepoints after damage induction. USP15-deficient cells typically show sustained γH2AX foci at 24h post-irradiation, indicating defective repair .

• Assess HR and NHEJ repair: Use integrated reporter assays to quantify HR and NHEJ repair efficiency. USP15 KO significantly compromises HR while minimally affecting NHEJ, confirming its specific role in the HR pathway .

• Test PARP inhibitor sensitivity: Assess sensitivity to PARP inhibitors (e.g., AZD2281) using colony formation assays. HR-deficient cells typically show hypersensitivity to PARP inhibition .

Include cell cycle analysis to rule out cell cycle effects on HR when interpreting results.

What approaches can I use to study USP15-protein interactions?

To investigate USP15 interactions with partner proteins (like BARD1), employ these complementary approaches:

• Co-Immunoprecipitation (Co-IP): Use antibodies against endogenous USP15 or BARD1 for reciprocal Co-IPs to detect native protein complexes. For example, studies have shown that endogenous USP15 and BARD1 associate with each other, and this interaction increases after DNA damage treatment .

• GST Pull-down Assays: Express and purify GST-tagged USP15 from E. coli and test its ability to pull down candidate interacting proteins from cell lysates or with purified proteins to confirm direct interactions .

• Domain Mapping: Create truncation mutants of both USP15 and its interacting partners to map the specific interaction regions. For example, USP15 deletion mutant (removing residues 740-981) abolishes binding with BARD1, while the BARD1 C-terminal BRCT domain (residues 568-777) is required for USP15 interaction .

• Functional Validation: Test the biological significance of identified interactions by expressing interaction-deficient mutants and assessing functional readouts such as HR efficiency or PARP inhibitor sensitivity .

• DNA Damage-Induced Interactions: Compare interaction strength before and after DNA damage induction using treatments like IR, HU, MMC, or CPT to assess damage-dependent complex formation .

How can I assess USP15 enzymatic activity in vitro?

To measure USP15 deubiquitinating activity, implement an in vitro deubiquitination assay:

• Protein Preparation:

  • Express and purify GST-fusion USP15 or His-tagged USP15 proteins from E. coli BL21 cells.

  • Prepare proteins in reaction buffer (50 mM Tris-HCl [pH 8.0], 50 mM NaCl, 1 mM EDTA, 10 mM DTT, 5% glycerol) .

• Substrate Preparation:

  • Generate ubiquitinated substrates (e.g., FLAG-BARD1-BRCT conjugated with HA-Ub) by expressing them in HEK293T cells.

  • Purify using FLAG M2 beads to isolate the ubiquitinated protein complex .

• Deubiquitination Reaction:

  • Incubate purified enzyme and substrate in reaction buffer at 37°C for 2 hours.

  • Analyze samples by Western blot using antibodies against the substrate and ubiquitin tags to detect deubiquitination .

• Controls and Variants:

  • Include catalytically inactive USP15 (C269A mutant) as a negative control.

  • Test reaction kinetics by collecting samples at different timepoints.

  • Include ubiquitin chain-specific antibodies to assess linkage-type specificity.

This assay allows for biochemical characterization of USP15's substrate specificity and enzymatic activity.

How does USP15 influence cancer cell response to PARP inhibitors?

USP15 critically regulates homologous recombination (HR) repair, which directly impacts cancer cell sensitivity to PARP inhibitors. Research data shows:

• USP15 knockout sensitizes cancer cells to PARP inhibitors: USP15-KO MCF7 cells show significantly reduced colony formation when treated with the PARP inhibitor AZD2281 compared to wild-type cells .

• Mechanism of action: USP15 interacts with BARD1 (a BRCA1 partner) via its C-terminal region, and this interaction increases after DNA damage. USP15 contributes to proper BARD1 recruitment to DNA double-strand breaks, which is essential for HR repair .

• Clinical implications: Cancer cells with reduced USP15 expression or function may exhibit "BRCAness" phenotype and respond better to PARP inhibitor therapy. This suggests USP15 status might serve as a biomarker for PARP inhibitor sensitivity .

• Experimental validation: When studying USP15's role in PARP inhibitor response, perform colony formation assays after treatment with varying concentrations of PARP inhibitors. Plate cells at low density, treat with the inhibitor, and culture for 14 days before staining colonies with crystal violet for quantification .

USP15 status may influence PARP inhibitor efficacy in clinical settings, making it a potential therapeutic target or predictive biomarker.

What is USP15's role in T cell activation and tumor immunity?

USP15 functions as a crucial negative regulator of T cell activation through a specific molecular mechanism:

• USP15 stabilizes MDM2: As a deubiquitinase, USP15 removes ubiquitin from MDM2 (an E3 ubiquitin ligase), preventing its degradation and thus stabilizing it .

• MDM2 targets NFATc2: The stabilized MDM2 negatively regulates T cell activation by targeting the transcription factor NFATc2 for degradation .

• USP15 deficiency enhances T cell responses: USP15-deficient T cells show enhanced activation in vitro and stronger responses to bacterial infection and tumor challenges in vivo .

• Dual anti-tumor effect: USP15 inhibition may provide dual benefits in cancer therapy by:

  • Inducing tumor cell apoptosis (through p53 pathway modulation)

  • Boosting anti-tumor T cell responses (through enhanced T cell activation)

This pathway represents a potential immunotherapeutic target, as inhibiting USP15 could potentially enhance anti-tumor immune responses while simultaneously promoting tumor cell death through p53-dependent mechanisms.

How can I study USP15 phosphorylation and its functional significance?

USP15 phosphorylation, particularly at Ser678, can be studied using these approaches:

• Phospho-specific antibody detection: Use phospho-USP15 (Ser678) antibodies for Western blot analysis. These can be custom-generated using phosphopeptide immunogens (e.g., "SENEN(pSER)QSEDSVGGC") .

• Antibody validation: Validate phospho-specific antibodies using:

  • Peptide blocking assays (pre-incubating antibody with phospho-peptide should eliminate signal)

  • Lambda phosphatase treatment of lysates (should eliminate signal)

  • Phosphomimetic (S678D) and phospho-dead (S678A) USP15 mutants as controls

• Kinase identification: Use kinase inhibitor panels and in vitro kinase assays to identify the kinase(s) responsible for USP15 Ser678 phosphorylation.

• Functional significance: Generate phospho-dead (S678A) and phosphomimetic (S678D) mutants of USP15 and compare:

  • Protein-protein interaction properties (especially with BARD1)

  • Localization to DNA damage sites

  • Deubiquitinating enzyme activity

  • HR efficiency and PARP inhibitor sensitivity

Phosphorylation may regulate USP15's activity, localization, or interaction with key partners in the DNA damage response pathway.

How can I optimize Western blot conditions for USP15 antibodies?

To achieve optimal Western blot results with USP15 antibodies, consider these technical recommendations:

• Sample preparation:

  • Use RIPA or NP-40 buffer with protease inhibitors and phosphatase inhibitors

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

  • Heat samples at 95°C for 5 minutes in SDS loading buffer

• Gel selection and transfer:

  • Use 8% gels to properly resolve USP15's 112 kDa band

  • Consider wet transfer for large proteins like USP15

  • Transfer at lower voltage (30V) overnight at 4°C for improved transfer efficiency

• Antibody dilution and incubation:

  • Start with recommended dilutions (e.g., 1:2000-1:12000 for 14354-1-AP)

  • Incubate primary antibody overnight at 4°C for best results

  • Use 5% BSA in TBST as blocking and antibody dilution buffer

• Controls and validation:

  • Include USP15 knockout/knockdown samples as negative controls

  • Use cell lines known to express USP15 as positive controls (HEK-293, HeLa, HepG2, K-562, MCF-7)

  • Consider running a panel of tissues (brain tissue shows good USP15 expression)

• Troubleshooting non-specific bands:

  • Increase washing time/frequency

  • Further dilute primary antibody

  • Verify antibody specificity with peptide competition or knockout controls

Expected result: A clean band at approximately 112 kDa corresponding to USP15's calculated molecular weight .

What are the optimal conditions for immunohistochemistry with USP15 antibodies?

For successful immunohistochemistry using USP15 antibodies, follow these recommendations:

• Tissue preparation and antigen retrieval:

  • Use formalin-fixed, paraffin-embedded (FFPE) tissues sectioned at 4-6 μm

  • Perform antigen retrieval using TE buffer pH 9.0 (preferred) or citrate buffer pH 6.0 as an alternative

  • Heat-induced epitope retrieval (pressure cooker or microwave) is recommended

• Antibody dilution and incubation:

  • Use 1:250-1:1000 dilution for primary antibody (optimization required for specific antibody)

  • Incubate primary antibody overnight at 4°C in a humidified chamber

  • Use appropriate secondary antibody detection system (HRP/DAB recommended)

• Controls and validation:

  • Include known positive tissues (placenta, breast cancer, lung, ovary tumor tissue show good USP15 expression)

  • Include antibody-omitted negative controls

  • Consider using tissues from USP15 knockout animals as specificity controls

• Signal amplification and counterstaining:

  • Consider tyramide signal amplification for weaker signals

  • Use hematoxylin for nuclear counterstaining (moderate intensity to avoid obscuring nuclear USP15)

  • Mounting with permanent mounting medium preserves signals

• Tissue-specific considerations:

  • For placenta: use brief proteinase K treatment before antigen retrieval

  • For brain tissue: extend antigen retrieval time

  • For lung tissue: additional blocking with avidin/biotin may reduce background

These conditions have been validated for detecting USP15 in human placenta, breast cancer, lung, ovary tumor, mouse kidney, and rat small intestine tissues .

How should I troubleshoot non-specific signals in immunofluorescence with USP15 antibodies?

When facing non-specific signals in immunofluorescence studies using USP15 antibodies, implement these troubleshooting strategies:

• Fixation optimization:

  • Test different fixation methods (4% PFA, methanol, or methanol-acetone)

  • Limit fixation time to 10-15 minutes at room temperature

  • For nuclear proteins, add a brief permeabilization step with 0.1% Triton X-100

• Blocking improvements:

  • Extend blocking time to 2 hours at room temperature

  • Use 5-10% normal serum from the species of secondary antibody

  • Add 0.3M glycine to blocking buffer to reduce aldehyde-induced background

  • Consider adding 0.1% Tween-20 to the blocking buffer

• Antibody dilution and incubation:

  • Dilute primary antibody 1:100-1:500 in blocking buffer

  • Incubate overnight at 4°C in a humidified chamber

  • Wash extensively (5x5 minutes) with PBS before and after secondary antibody

• Controls and validation:

  • Include antibody pre-absorption controls using the immunizing peptide

  • Use USP15 knockout or knockdown cells as negative controls

  • Perform secondary-only controls to assess non-specific binding

  • Test multiple USP15 antibodies targeting different epitopes for confirmation

• Image acquisition optimization:

  • Adjust exposure settings based on negative controls

  • Use sequential scanning for multi-channel imaging to prevent bleed-through

  • Consider using spectral unmixing for overlapping fluorophores

These strategies should help distinguish specific USP15 signals from non-specific background in immunofluorescence applications.

How can I design experiments to study USP15's dual role in cancer?

To investigate USP15's complex roles in both cancer cell survival and anti-tumor immunity, design these complementary experimental approaches:

• Cancer cell-intrinsic effects:

  • Generate USP15 knockdown or knockout cancer cell lines using shRNA or CRISPR-Cas9

  • Assess p53 stabilization, MDM2 levels, and apoptotic markers

  • Measure colony formation and proliferation rates

  • Analyze DNA damage response and repair efficiency

  • Test sensitivity to chemotherapeutics and PARP inhibitors

• Immune cell effects:

  • Generate T cell-specific USP15 knockout mice (using Cd4-Cre or similar)

  • Assess T cell activation markers ex vivo

  • Measure NFATc2 protein levels and MDM2 stabilization

  • Conduct T cell proliferation and cytokine production assays

  • Perform adoptive transfer experiments with USP15-deficient T cells

• Integrated tumor models:

  • Implant syngeneic tumors in USP15 wild-type vs. knockout mice

  • Create bone marrow chimeras to distinguish immune vs. tumor cell effects

  • Analyze tumor-infiltrating lymphocytes for activation status

  • Combine USP15 inhibition with checkpoint blockade therapy

  • Monitor cancer cell death mechanisms in vivo

• Therapeutic targeting validation:

  • Test small molecule USP15 inhibitors on cancer cells and T cells separately

  • Assess efficacy in immunocompetent vs. immunodeficient tumor models

  • Analyze pharmacodynamic markers (MDM2 stability, NFATc2 levels)

  • Evaluate combination therapies with existing cancer treatments

This comprehensive approach will help dissect USP15's multifaceted roles in cancer biology and identify optimal targeting strategies.

What are the in vivo phenotypes of USP15 deficiency relevant to cancer research?

USP15 deficiency produces distinct in vivo phenotypes that have important implications for cancer research:

• Genomic instability:

  • USP15-deficient (−/− Usp15) mouse embryonic fibroblasts (MEFs) exhibit a nearly 3-fold increase in spontaneous single chromatid breaks compared to wild-type MEFs, indicating intrinsic genomic instability .

  • This suggests that USP15 functions as a genome maintenance factor under normal conditions.

• Radiation sensitivity:

  • USP15-deficient mice show increased sensitivity to whole-body irradiation, with only 10% of homozygous knockout mice surviving 1 month after irradiation compared to 45% survival in wild-type mice .

  • Tissues from USP15-deficient mice show elevated γH2AX signaling, indicating persistent DNA damage .

• Cellular DNA repair defects:

  • MEFs from USP15-deficient mice exhibit defects in BARD1, RPA, and RAD51 foci formation following ionizing radiation .

  • These cells also show hypersensitivity to DNA-damaging agents including camptothecin, mitomycin C, and PARP inhibitors like AZD2281 .

• Enhanced anti-tumor immunity:

  • USP15 deficiency promotes T cell activation and enhances T cell responses to bacterial infection and tumor challenge in vivo .

  • This suggests that USP15 inhibition might boost anti-tumor immune responses in addition to directly affecting cancer cell survival.

These findings highlight the potential value of USP15 as a therapeutic target in cancer, as its inhibition may simultaneously sensitize cancer cells to DNA-damaging treatments while enhancing anti-tumor immunity.

How can in vitro models best recapitulate USP15's physiological functions?

To create in vitro experimental systems that accurately represent USP15's physiological functions, consider these methodological approaches:

• Cell line selection:

  • Use multiple cell lines to account for tissue-specific effects

  • Include both cancer (HEK-293, HeLa, HepG2, K-562, MCF-7) and normal cells (fibroblasts, primary T cells)

  • When possible, use primary cells derived from USP15-deficient mice alongside wild-type controls

• Physiological protein levels:

  • Avoid overexpression systems when studying USP15 function

  • Use CRISPR-Cas9 to tag endogenous USP15 rather than ectopic expression

  • For rescue experiments, titrate expression to match endogenous levels

• DNA damage induction:

  • Use clinically relevant doses of DNA-damaging agents

  • Apply low-dose ionizing radiation (1-2 Gy) to mimic therapeutic exposures

  • Use diverse DNA damage inducers to assess pathway specificity (CPT, MMC, HU, IR)

• Immune context models:

  • For T cell studies, use physiological T cell receptor stimulation

  • Consider co-culture systems with antigen-presenting cells

  • Use primary T cells from USP15-deficient mice to avoid artifacts of knockdown

• 3D culture systems:

  • Implement organoid cultures for epithelial studies

  • Use spheroid cultures for cancer studies

  • Consider extracellular matrix components when studying DNA damage responses

• Time-resolved experiments:

  • Track USP15 function over physiologically relevant timeframes

  • For DNA damage studies, examine both early (0-1h) and late (24h) responses

  • For T cell activation, follow response kinetics from minutes to days

These approaches will help ensure that in vitro findings accurately reflect USP15's complex roles in vivo, providing more translationally relevant insights.