USP17L6P Antibody

What is USP17L6P and what cellular functions does it regulate?

USP17L6P (Q6QN14) is a deubiquitinating enzyme that removes conjugated ubiquitin from specific proteins to regulate several cellular processes including cell proliferation, progression through the cell cycle, cell migration, and cellular responses to viral infection. Research indicates that while it actively participates in these processes, it appears to be non-functional in apoptosis regulation .

USP17L6P belongs to the DUB/USP17 subfamily, originally identified as cytokine-inducible immediate early genes. Members of this family have been implicated in cell growth and survival regulation, with evidence showing that constitutive expression of human USP17 can block cell proliferation .

What types of USP17L6P antibodies are commercially available for research?

Several types of USP17L6P antibodies are available for research applications:

These antibodies typically target amino acids 136-398 at the C-terminal region of the protein and are generated using recombinant human USP17L6P protein as the immunogen .

How should USP17L6P antibodies be stored to maintain their activity?

For optimal preservation of antibody activity, USP17L6P antibodies should be stored at -20°C or -80°C upon receipt. They are typically provided in a liquid formulation containing preservatives (0.03% Proclin 300) and stabilizers (50% Glycerol in 0.01M PBS, pH 7.4). Repeated freeze-thaw cycles should be avoided to prevent degradation of the antibody .

For long-term storage, it is advisable to aliquot the antibody into smaller volumes before freezing to minimize the number of freeze-thaw cycles. When handled properly, these antibodies can maintain their activity for extended periods, enabling consistent experimental results.

What are the validated applications for USP17L6P antibodies?

USP17L6P antibodies have been validated for the following applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): All forms (unconjugated, HRP-conjugated, and biotin-conjugated) are suitable for ELISA applications .

• Western Blotting (WB): Primarily the unconjugated form, with recommended dilutions ranging from 1:1000 to 1:5000 .

The choice of application depends on the specific research question. ELISA is typically used for quantitative detection of USP17L6P in samples, while Western blotting allows for size determination and semi-quantitative analysis of the protein.

How can I optimize Western blotting protocols when using USP17L6P antibodies?

For optimal Western blotting results with USP17L6P antibodies:

• Sample preparation: Use appropriate lysis buffers containing protease inhibitors to prevent degradation of USP17L6P.

• Protein loading: Load 20-50 μg of total protein per lane.

• Antibody dilution: Start with a 1:1000 dilution and adjust as needed based on signal strength. The recommended range is 1:1000-1:5000 .

• Incubation conditions: Incubate with primary antibody overnight at 4°C for best results.

• Detection system: Choose a detection system compatible with rabbit IgG. For HRP-conjugated antibodies, use chemiluminescent substrates; for unconjugated antibodies, use appropriate secondary antibodies.

• Controls: Include positive controls (tissues or cells known to express USP17L6P) and negative controls (tissues or cells with low or no expression) to validate specificity.

What experimental approaches can be used to study USP17L6P's deubiquitinating activity?

To investigate USP17L6P's deubiquitinating activity, researchers can employ several methodological approaches:

• Co-immunoprecipitation assays: To detect interactions between USP17L6P and potential substrate proteins. This approach was successfully used to demonstrate that the related USP17 interacts with and deubiquitinates RCE1 .

• In vitro deubiquitination assays: Using purified USP17L6P and ubiquitinated substrates to directly measure removal of ubiquitin chains.

• Cell-based ubiquitination assays: Co-expressing USP17L6P with tagged ubiquitin and potential substrate proteins, followed by immunoprecipitation and Western blotting to detect changes in ubiquitination levels.

• Active site mutants: Creating catalytically inactive mutants (similar to USP17CS) for comparison with wild-type enzyme to confirm deubiquitinating activity .

• RNAi approaches: Using shRNA targeting USP17L6P to assess the effects of its knockdown on substrate ubiquitination levels and downstream cellular processes .

How does USP17L6P interact with the Ras signaling pathway?

Based on studies with the related family member USP17, there appears to be significant interplay between USP17 family deubiquitinases and Ras signaling:

• RCE1 deubiquitination: USP17 specifically deubiquitinates RCE1 (Ras-converting enzyme 1), a key enzyme involved in Ras processing .

• Effect on RCE1 activity: Deubiquitination by USP17 reduces RCE1's catalytic activity, thereby affecting Ras processing .

• Ras membrane localization: Expression of USP17 blocks proper localization of Ras to the plasma membrane, which is essential for Ras function .

• Downstream signaling effects: USP17 expression decreases activation of MEK and ERK, key downstream components of the Ras signaling pathway .

• Cell proliferation: The inhibitory effect of USP17 on cell proliferation is dependent on RCE1, as USP17 cannot block proliferation in RCE1-null cells .

Research investigating whether USP17L6P specifically shares these functions is ongoing, but the high degree of homology suggests similar mechanisms may be involved.

What is the subcellular localization of USP17L6P and how does it relate to function?

Studies with the USP17 family suggest these deubiquitinating enzymes localize primarily to the endoplasmic reticulum (ER):

• ER localization: USP17 shows perinuclear distribution consistent with ER localization, which has been confirmed by co-localization with the ER marker calnexin .

• Co-localization with substrates: USP17 co-localizes with RCE1 at the ER, positioning it appropriately to regulate RCE1 activity .

• Functional significance: This localization is functionally important, as it places USP17 in proximity to the Ras processing machinery, which also occurs at the ER.

• Catalytically inactive mutants: Interestingly, catalytically inactive USP17CS shows more pronounced perinuclear distribution, suggesting that loss of catalytic activity may trap the enzyme at the ER .

These localization patterns provide important insights into the functional mechanisms of USP17 family proteins, potentially including USP17L6P.

How do USP17L6P's functions compare with other USP17 family members?

The USP17 family includes multiple members with varying degrees of sequence similarity and potentially overlapping functions:

• Sequence homology: USP17L6P shares significant sequence homology with other family members including USP17C, USP17D, and USP17N, which are often listed as synonyms .

• Cytokine regulation: Several family members, including USP17 (DUB-3), are cytokine-inducible immediate early genes, suggesting shared regulatory mechanisms .

• Cell cycle effects: Multiple family members, including DUB-1, can induce cell cycle arrest prior to S phase when overexpressed .

• Apoptosis regulation: While some family members like DUB-2 can inhibit apoptosis induced by cytokine withdrawal, USP17L6P appears to be non-functional in apoptosis regulation .

• Ras pathway regulation: USP17 has been shown to regulate Ras signaling through RCE1 deubiquitination; similar functions may exist for USP17L6P but require further investigation .

What are common pitfalls when working with USP17L6P antibodies and how can they be addressed?

Researchers may encounter several challenges when working with USP17L6P antibodies:

• Cross-reactivity: Due to high sequence homology between USP17 family members, antibodies may cross-react with related proteins. Solution: Validate specificity using knockout/knockdown cells or peptide competition assays.

• Low expression levels: USP17L6P may be expressed at low levels in many cell types. Solution: Use enrichment techniques like immunoprecipitation before Western blotting or highly sensitive detection systems.

• Buffer incompatibility: Some buffer components may interfere with antibody binding. Solution: Ensure compatibility between sample buffers and antibody dilution buffers; consider dialysis if necessary.

• Storage degradation: Antibody activity may decrease with improper storage. Solution: Store at recommended temperatures (-20°C or -80°C), avoid freeze-thaw cycles by preparing aliquots .

• Batch variability: Different lots may show slight variations in performance. Solution: Test each new lot against a reference sample to establish appropriate working dilutions.

How can USP17L6P expression and activity be modulated for functional studies?

To effectively study USP17L6P function, researchers can modulate its expression and activity using several approaches:

• RNA interference: shRNA or siRNA targeting USP17L6P can effectively knock down expression, as demonstrated with USP17 .

• Overexpression systems: Transfection with USP17L6P expression constructs can be used to study gain-of-function effects.

• Catalytically inactive mutants: Creating catalytic site mutants (similar to USP17CS) provides important controls for distinguishing between deubiquitinating-dependent and independent functions .

• Cytokine stimulation: Given that related family members are cytokine-inducible, treatment with specific cytokines may upregulate endogenous USP17L6P .

• CRISPR-Cas9 gene editing: For complete knockout studies, CRISPR-Cas9 can be employed to generate USP17L6P-null cell lines.

How can I determine if USP17L6P is involved in the deubiquitination of my protein of interest?

To investigate whether USP17L6P deubiquitinates a specific protein of interest:

• Co-immunoprecipitation: Determine if USP17L6P physically interacts with your protein of interest using co-immunoprecipitation with USP17L6P antibodies.

• Ubiquitination assays: Express HA-tagged ubiquitin and your protein of interest with or without USP17L6P, then immunoprecipitate your protein and blot for ubiquitin to detect changes in ubiquitination levels .

• Catalytic mutant controls: Include a catalytically inactive USP17L6P mutant as a control to confirm that any observed deubiquitination is dependent on enzymatic activity .

• In vitro deubiquitination: Perform in vitro assays with purified components to directly assess USP17L6P's ability to remove ubiquitin from your protein.

• Linkage-specific analysis: Determine which ubiquitin chain types (K48, K63, etc.) are affected by USP17L6P, as this provides insight into functional consequences. For example, USP17 specifically removes K63-linked chains from RCE1 .

How might USP17L6P antibodies be utilized in cancer research?

USP17L6P antibodies can be valuable tools in cancer research:

• Expression analysis: Determining USP17L6P expression levels in different cancer types using immunohistochemistry or Western blotting.

• Prognostic marker investigation: Correlating USP17L6P expression with clinical outcomes to assess its potential as a prognostic marker.

• Ras pathway dysregulation: Given the relationship between USP17 family members and Ras signaling, USP17L6P antibodies can help investigate potential roles in cancers with Ras pathway dysregulation .

• Therapeutic target validation: Assessing USP17L6P as a potential therapeutic target, particularly if it shares USP17's ability to regulate cell proliferation .

• Drug development: Developing antibody-based approaches to modulate USP17L6P activity or using antibodies to assess the efficacy of small molecule inhibitors targeting USP17L6P.

What is known about the relationship between USP17L6P and other deubiquitinating enzymes in immune modulation?

While specific information about USP17L6P in immune modulation is limited, insights from related deubiquitinating enzymes suggest potential roles:

• USP7 parallels: USP7, another deubiquitinating enzyme, has been shown to interact with and stabilize PD-L1, affecting immune checkpoint regulation in cancer .

• Cytokine regulation: Like other USP17 family members, USP17L6P may be regulated by cytokines, suggesting involvement in immune response pathways .

• Viral response: USP17L6P is implicated in cellular responses to viral infection, indicating a potential role in antiviral immunity .

• Cell migration effects: Through its effects on cell migration, USP17L6P may influence immune cell trafficking and function.

• DUB redundancy: Functional redundancy with other deubiquitinating enzymes may exist in immune pathways, complicating the assessment of USP17L6P-specific effects.

How can quantitative proteomics be integrated with USP17L6P antibody-based techniques for comprehensive substrate identification?

Advanced integrated approaches combining proteomics with USP17L6P antibody techniques can facilitate comprehensive substrate identification:

• Immunoprecipitation-mass spectrometry (IP-MS): Using USP17L6P antibodies to immunoprecipitate the enzyme along with interacting proteins, followed by mass spectrometry identification.

• Ubiquitin remnant profiling: Combining USP17L6P manipulation (overexpression or knockdown) with proteome-wide identification of ubiquitinated lysines using ubiquitin remnant antibodies and mass spectrometry.

• SILAC or TMT labeling: Implementing quantitative proteomics approaches with stable isotope labeling to compare changes in the ubiquitinome upon USP17L6P modulation.

• Proximity labeling: Using BioID or APEX2 fusions with USP17L6P to identify proteins in close proximity, potentially including substrates.

• Correlation analysis: Correlating changes in USP17L6P expression across multiple samples with changes in ubiquitination levels of potential substrates to identify regulated targets.

These integrated approaches can provide comprehensive and unbiased identification of USP17L6P substrates, advancing our understanding of its cellular functions.