USP36 Antibody

What is USP36 and what are its primary cellular functions?

USP36, also known as KIAA1453, FLJ12851, or DUB1, is a member of the peptidase C19 family of deubiquitinating enzymes. It functions primarily as a deubiquitinase that regulates transcription and processing of rRNA by removing ubiquitin from various proteins involved in these processes . Recent research has established several critical functions:

• Histone H2B deubiquitination: USP36 interacts with H2B and removes monoubiquitin from H2Bub1 both in cells and in vitro, affecting gene expression regulation .

• Nucleolar activity: USP36 is exclusively located in the nucleolus and plays essential roles in ribosome biogenesis .

• Cell proliferation regulation: Knockdown of USP36 induces p21 expression and significantly inhibits cell proliferation .

• Cancer progression: USP36 is highly expressed in breast cancer tissues and cell lines, with high expression predicting poor prognosis in patients .

What applications are USP36 antibodies most suitable for in research settings?

Based on validated applications, USP36 antibodies are suitable for multiple experimental approaches:

Beyond these applications, USP36 antibodies have been used successfully for chromatin immunoprecipitation (ChIP) studies examining the role of USP36 in regulating gene expression at specific loci, such as the p21 gene .

How should researchers optimize Western blot protocols for USP36 detection?

When detecting USP36 via Western blot, consider these important optimization factors:

• Expected molecular weight considerations: While the calculated molecular weight of USP36 is 123 kDa (1123 amino acids), the observed molecular weight is approximately 150 kDa . This discrepancy is likely due to post-translational modifications.

• Sample preparation: For optimal USP36 detection, lyse cells in RIPA buffer containing protease inhibitors. Sonication on ice may improve extraction, especially for nucleolar proteins .

• Blocking and antibody incubation: Use 5% non-fat milk or BSA in TBST for blocking. For primary antibody incubation, dilute USP36 antibody between 1:2000-1:10000 depending on the sample type and antibody lot .

• Controls: Include positive control lysates from cells known to express USP36 (e.g., HeLa, U2OS, or Jurkat cells) and consider using USP36 knockdown cells as negative controls to validate specificity .

What are best practices for immunoprecipitation experiments examining USP36 interactions?

For effective immunoprecipitation of USP36 and its binding partners:

• Lysis conditions: Use 1% SDS-containing RIPA buffer with sonication on ice for complete lysis, especially when studying nuclear proteins. For analyzing protein interactions, consider milder detergents to preserve protein-protein interactions .

• Pre-clearing: Treat lysates with Protein A/G Plus-Agarose for 1 hour to reduce non-specific binding .

• Antibody amount: Use 0.5-4.0 μg of USP36 antibody per 1.0-3.0 mg of total protein lysate .

• Salt concentration consideration: For studying stable protein complexes like USP36-Snail1, interactions can be detected even in the presence of 500 mM NaCl, indicating very strong binding .

• Controls: Always include IgG control immunoprecipitations to identify non-specific binding .

How can researchers effectively use USP36 antibodies in immunofluorescence studies?

For optimal immunofluorescence detection of USP36:

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature, as USP36 is sensitive to fixation conditions.

• Permeabilization: Since USP36 is predominantly nucleolar, ensure complete nuclear permeabilization using 0.2% Triton X-100.

• Antibody dilution: Use USP36 antibody at 1:400-1:1600 dilution for immunofluorescence applications .

• Co-localization studies: For examining nucleolar localization, co-stain with established nucleolar markers. Under normal conditions, USP36 is exclusively located in nucleoli, while proteins like Snail1 relocalize to nucleoli under ribotoxic stress .

• Confocal microscopy: High-resolution confocal microscopy is recommended for accurate visualization of nucleolar structures and co-localization.

How can USP36 antibodies be used to study histone deubiquitination mechanisms?

Researchers investigating USP36's role in histone deubiquitination should consider these approaches:

• In vitro deubiquitination assays: Using purified USP36 (wild-type and catalytic mutant C131A) and ubiquitinated histones to directly assess enzymatic activity .

• ChIP-sequencing: To map genome-wide distribution of USP36 and correlate with H2Bub1 patterns, p21 has been established as a model gene for studying USP36's role in histone H2B deubiquitination .

• ChIP-qPCR: For targeted analysis of specific loci, such as the p21 gene body where H2Bub1 is enriched. Design primers for promoter, gene body, and 3' regions to capture the distribution pattern .

• Sequential ChIP: To determine if USP36 and H2Bub1 occupy the same genomic regions but are mutually exclusive due to the deubiquitinating activity.

• Functional validation: Compare wild-type USP36 with the catalytically inactive C131A mutant to confirm that the deubiquitination function is responsible for observed effects .

What methodological approaches should be used to investigate USP36's role in cancer progression?

For researchers examining USP36's contributions to cancer:

How can researchers examine the interplay between USP36 and cellular stress responses?

To investigate USP36's role in stress response pathways:

• Ribotoxic stress induction: Treat cells with ribotoxic stress inducers like homoharringtonine (HHT), puromycin, anisomycin, or blasticidin, which significantly upregulate both USP36 and its substrate proteins like Snail1 .

• Nucleolar stress visualization: Perform immunofluorescence co-localization studies to track USP36 and its interaction partners (e.g., Snail1) under normal and stress conditions. Under ribotoxic stress, proteins like Snail1 relocalize from the nucleoplasm to nucleoli where they co-localize with USP36 .

• Protein stability assays: Compare protein half-life of USP36 substrates between normal and stress conditions using cycloheximide chase assays. Wild-type USP36, but not the catalytically inactive C131A mutant, significantly extends the half-life of substrate proteins .

• Nucleolar isolation: For biochemical analysis of stress-induced changes in nucleolar composition and USP36 interactions, perform nucleolar isolation followed by immunoprecipitation and mass spectrometry.

What protocols are recommended for studying USP36-mediated deubiquitination of target proteins?

For investigating USP36's deubiquitinating activity:

• Ubiquitination assays: Lyse cells in 1% SDS-containing RIPA buffer with sonication on ice. Pre-clear lysates with Protein A/G Plus-Agarose for 1 hour, then immunoprecipitate with appropriate antibodies overnight at 4°C. Wash precipitates four times with Protein A/G Plus-Agarose beads .

• Detection methods:

  • Immunoblot with anti-ubiquitin antibody to detect total ubiquitination levels

  • Immunoblot with substrate-specific antibodies (e.g., anti-PKM2, anti-H2B) to detect specific targets

• Controls and comparisons:

  • Compare wild-type USP36 with catalytically inactive C131A mutant

  • Include proteasome inhibitor treatment (e.g., MG132) as a positive control for ubiquitinated protein accumulation

  • For verification of specific substrates, perform in vitro deubiquitination assays with purified components

• Substrate validation: For newly identified substrates, verify direct interaction through co-immunoprecipitation under high salt conditions (e.g., 500 mM NaCl) to ensure specificity .

How can researchers identify and validate novel USP36 substrates?

For discovering new USP36 substrates:

• Proteomic screening approaches:

  • Compare ubiquitinated protein profiles between control and USP36-depleted cells using mass spectrometry

  • Perform USP36 immunoprecipitation followed by mass spectrometry to identify interacting proteins

  • Use proximity labeling approaches (BioID or TurboID) to identify proteins in close proximity to USP36 in nucleoli

• Validation experiments:

  • Co-immunoprecipitation to confirm physical interaction

  • In vitro and in vivo deubiquitination assays

  • Protein stability assays (cycloheximide chase) with and without USP36

  • Mutational analysis of potential ubiquitination sites on substrate proteins

• Functional relationship validation: Study the functional consequences of the USP36-substrate relationship through genetic manipulation (e.g., overexpression of wild-type or mutant USP36) and phenotypic assays relevant to the substrate's known functions .

What explains the discrepancy between calculated and observed molecular weights for USP36?

The calculated molecular weight of USP36 is 123 kDa (1123 amino acids), but the observed molecular weight in SDS-PAGE is approximately 150 kDa . This discrepancy may be attributed to:

• Post-translational modifications: USP36 may undergo phosphorylation, SUMOylation, or other modifications that affect electrophoretic mobility.

• Protein structure considerations: Some proteins migrate aberrantly due to their amino acid composition or structural features.

• Incomplete denaturation: Residual tertiary structure can affect migration patterns.

When interpreting Western blot results:

• Always include positive control lysates from cells known to express USP36

• Consider that different isoforms may exist in different cell types

• Be aware that post-translational modifications may vary under different experimental conditions or cell states

What are common challenges and solutions when using USP36 antibodies in research?

How are USP36 antibodies being used to explore potential therapeutic interventions?

Recent research suggests several promising therapeutic applications:

• Cancer therapeutic development: High USP36 expression predicts poor prognosis in breast cancer patients, suggesting its potential as both a biomarker and therapeutic target . Researchers can use USP36 antibodies to:

  • Screen for compounds that modulate USP36 expression or activity

  • Evaluate the effects of USP36 inhibition on cancer cell viability and metabolism

  • Monitor changes in USP36 levels during treatment response

• miRNA-based therapies: The miR-140-3p/USP36/PKM2 axis has been identified as a regulatory pathway in breast cancer . Researchers investigating miRNA-based interventions can use USP36 antibodies to:

  • Validate miRNA targeting efficiency

  • Monitor USP36 protein levels following miRNA treatment

  • Assess downstream effects on ubiquitination of target proteins like PKM2

• Metabolic pathway intervention: Inhibiting USP36 significantly reduces ATP production, lactate and pyruvate levels, and glucose uptake in cancer cells . Researchers investigating metabolic interventions can use USP36 antibodies to:

  • Correlate USP36 levels with metabolic parameters

  • Evaluate the efficacy of combination therapies targeting both USP36 and metabolic enzymes

What methods are recommended for studying USP36's role in ribosome biogenesis?

For researchers investigating USP36's nucleolar functions:

• Nucleolar protein extraction: Use specialized protocols for isolating nucleolar proteins with high purity.

• Ribosome biogenesis assays: Monitor pre-rRNA processing and mature rRNA production in cells with modulated USP36 levels.

• Co-localization studies: Perform immunofluorescence to examine USP36 localization with other nucleolar markers under different conditions.

• Stress response analysis: Investigate how ribotoxic stress affects USP36 expression and function in the nucleolus. Ribotoxic stress inducers like HHT, puromycin, anisomycin, and blasticidin significantly upregulate USP36 protein expression .