USP4 Antibody

What is USP4 and why is it significant in molecular research?

USP4 (ubiquitin-specific peptidase 4) functions as a deubiquitinating enzyme that removes ubiquitin moieties from target proteins, preventing their degradation and stabilizing key signaling molecules. This protein plays crucial roles in signal transduction, transcriptional regulation, and cell cycle progression by modulating the activity and availability of proteins critical for these pathways . USP4 has garnered significant attention due to its interaction with various cellular components, including the spliceosome machinery which impacts RNA processing and gene expression . Additionally, USP4 interacts with the C-terminus of the Adenosine A2A receptor, enhancing cell surface expression and functional activity, highlighting its importance in receptor signaling and potential implications for therapeutic development .

How do I select the appropriate USP4 antibody for my experimental needs?

Selection of an appropriate USP4 antibody should be guided by several experimental considerations:

• Experimental application: Different applications require different antibody formats and properties. For western blotting, both polyclonal and monoclonal antibodies can be used, with recommended dilutions typically between 1:500-1:2000 . For immunohistochemistry, appropriate dilutions range from 1:200-1:800 .

• Species reactivity: Ensure the antibody recognizes USP4 in your model organism. Available antibodies demonstrate reactivity with human, mouse, and rat USP4 .

• Antibody type: Consider whether a monoclonal (higher specificity) or polyclonal (typically greater sensitivity) antibody is appropriate for your needs. For instance, the H-3 mouse monoclonal IgG1 antibody detects USP4 across multiple species and applications , while rabbit polyclonal antibodies like 24976-1-AP target human USP4 in various applications .

• Conjugation requirements: USP4 antibodies are available in non-conjugated forms and various conjugated forms including agarose, horseradish peroxidase (HRP), phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates, which should be selected based on your detection system .

What are the molecular characteristics of USP4 that influence antibody binding and detection?

USP4 has several characteristics that researchers should consider when designing experiments:

• Molecular weight: USP4 has a calculated molecular weight of 109 kDa (963 amino acids) , but exists in 3 isoforms with molecular masses of 36, 104, and 109 kDa . This variation necessitates careful interpretation of western blot results.

• Protein domains: USP4 belongs to the peptidase C19 family and USP4 subfamily . Antibodies targeting different domains may yield different results in assays dependent on protein conformation.

• Post-translational modifications: As a deubiquitinating enzyme, USP4's function involves interaction with ubiquitinated proteins, which could affect epitope accessibility in certain experimental conditions.

• Subcellular localization: USP4's distribution within cells may require specific sample preparation protocols to ensure proper antigen retrieval and accessibility, particularly for immunohistochemistry applications .

What are the optimal conditions for using USP4 antibodies in Western blotting applications?

For optimal Western blotting results with USP4 antibodies, researchers should consider:

• Sample preparation: Complete cell lysis is essential for proper detection of USP4. THP-1 cells have been validated as positive controls for Western blotting with USP4 antibodies .

• Antibody dilution: For polyclonal antibodies like 24976-1-AP, a dilution range of 1:500-1:2000 is recommended . The precise dilution should be titrated in each testing system to obtain optimal results.

• Expected molecular weight: USP4 typically appears at 109 kDa, but researchers should be aware of the three isoforms (36, 104, and 109 kDa) that might appear depending on the cell type and experimental conditions .

• Detection systems: When using HRP-conjugated secondary antibodies or direct HRP-conjugated USP4 antibodies (e.g., sc-376000 HRP), ensure compatible chemiluminescent substrates for optimal signal-to-noise ratios .

• Controls: Include positive controls (THP-1 cells) and negative controls (lysates from cells with USP4 knockdown) to validate antibody specificity.

How can USP4 antibodies be effectively utilized in immunohistochemistry studies?

Effective immunohistochemistry with USP4 antibodies requires attention to several critical parameters:

• Tissue preparation: Proper fixation and embedding are essential. Human ovary cancer tissue has been validated for USP4 immunohistochemistry .

• Antigen retrieval: For optimal results, antigen retrieval with TE buffer pH 9.0 is suggested. Alternatively, citrate buffer pH 6.0 may be used, though comparative effectiveness should be determined empirically for specific tissue types .

• Antibody dilution: A recommended dilution range of 1:200-1:800 for immunohistochemistry applications provides a starting point, though optimization for specific tissues is advised .

• Detection systems: The choice between DAB (3,3'-diaminobenzidine) and AEC (3-amino-9-ethylcarbazole) chromogens should be based on the desired signal stability and counterstaining requirements.

• Controls: Include positive control tissues with known USP4 expression and negative controls (primary antibody omission or isotype controls) to validate staining specificity.

What approaches can be used to validate USP4 antibody specificity in experimental systems?

Validating USP4 antibody specificity is crucial for generating reliable data. Multiple approaches should be employed:

• Western blot analysis: Compare the molecular weight of detected bands with the expected size of USP4 (109 kDa). Multiple bands at 36, 104, and 109 kDa may represent different isoforms .

• Genetic knockdown/knockout validation: Use shRNA or CRISPR-Cas9 to reduce USP4 expression and confirm reduced antibody signal. For example, shUSP4#2 and shUSP4#3 have been used to successfully knockdown USP4 in KYSE150 cells, with verification by both mRNA and protein level analyses .

• Overexpression controls: Complement knockdown studies with USP4 overexpression, as demonstrated in KYSE180 cells where USP4 overexpression significantly increased both mRNA and protein levels .

• Immunoprecipitation followed by mass spectrometry: This approach can confirm that the antibody is precipitating the correct protein by peptide identification.

• Multiple antibody verification: Use different antibodies targeting distinct epitopes of USP4 to confirm consistent localization and expression patterns.

How does USP4 contribute to cancer progression, and how can this be studied using USP4 antibodies?

USP4 has been implicated in multiple cancer types, and USP4 antibodies are valuable tools for investigating its role:

• Esophageal squamous cell carcinoma (ESCC): USP4 expression is markedly upregulated in ESCC tumor tissues and cells. Loss- and gain-of-function assays have demonstrated that USP4 silencing inhibits ESCC cell proliferation, migration, and invasion, while USP4 overexpression promotes these behaviors . USP4 antibodies can be used in:

  a) Immunohistochemistry: To compare USP4 expression between tumor and adjacent normal tissues

  b) Western blotting: To quantify USP4 protein levels after experimental manipulation

  c) Immunoprecipitation: To identify USP4 interaction partners in cancer cells

• Lung cancer: USP4 has been reported as a critical factor in promoting lung cancer stemness and potentially serves as a useful lung cancer prognosis marker . Antibody-based studies can help:

  a) Evaluate USP4 expression in patient samples: Correlating expression with clinical outcomes

  b) Investigate cellular localization: Determining if USP4 redistribution occurs in tumor versus normal cells

  c) Identify downstream effectors: Through co-immunoprecipitation studies followed by proteomics

• Experimental design considerations: When studying USP4 in cancer models, researchers should implement:

  a) Multiple cancer cell lines: To account for heterogeneity

  b) In vivo models: USP4 silencing has been shown to repress tumor growth and metastasis in ESCC nude mouse models

  c) Correlation with clinical parameters: Stage, grade, metastasis, and patient survival

What is the role of USP4 in p53 regulation and how can this be experimentally investigated?

USP4 plays a significant role in regulating p53, a key tumor suppressor protein:

• Mechanism of action: USP4 inhibits p53 through deubiquitinating and stabilizing ARF-BP1, a p53-targeting E3 ubiquitin ligase . This represents an indirect mechanism of p53 regulation.

• Experimental approaches:

  a) Protein stability assays: Overexpression of USP4 in U2OS cells significantly reduces p53 levels compared to control cells, particularly following DNA damage (NCS treatment)

  b) siRNA knockdown studies: Inhibiting USP4 by specific siRNAs results in higher levels of p53, suggesting USP4 inhibits p53 stabilization

  c) Co-immunoprecipitation: Can be used to confirm USP4 interaction with ARF-BP1

  d) Ubiquitination assays: To demonstrate USP4's deubiquitinating activity on ARF-BP1

  e) Cell line selection: Isogenic HCT116 cell lines can be employed to study USP4 effects in different p53 backgrounds

• Technical considerations:

  a) Timing of analysis: p53 levels typically increase within 2 hours after DNA damage treatment, reaching peak levels after approximately 8 hours

  b) Controls: Include both overexpression and knockdown conditions to establish causality

  c) Multiple p53 readouts: Examine both p53 protein levels and transcriptional activity of p53 target genes

How can USP4 antibodies be used to investigate the interaction between USP4 and the TGF-β signaling pathway?

The interaction between USP4 and TGF-β signaling, particularly through TAK1 (Transforming growth factor-β-activated kinase 1), represents an important area of research:

• Established relationship: USP4 specifically interacts with TAK1 and stabilizes TAK1 protein levels via deubiquitination in ESCC cells . This interaction appears crucial for ESCC progression.

• Experimental approaches:

  a) Co-immunoprecipitation: USP4 antibodies can be used to pull down USP4 and detect co-precipitating TAK1, confirming their physical interaction

  b) Proximity ligation assay: To visualize and quantify USP4-TAK1 interactions in situ within cells

  c) Ubiquitination assays: To demonstrate USP4's deubiquitinating activity on TAK1

  d) siRNA-mediated knockdown: To examine how USP4 depletion affects TAK1 protein stability and downstream signaling

• Functional readouts:

  a) Proliferation assays: Colony formation, CCK-8, and EdU incorporation assays have demonstrated that USP4 knockdown inhibits proliferation while USP4 overexpression promotes proliferation

  b) Migration and invasion assays: To assess how modulating the USP4-TAK1 axis affects these cancer-relevant phenotypes

  c) In vivo models: Tumor growth and metastasis can be evaluated in nude mouse models with manipulated USP4 expression

How can USP4 antibodies be utilized in multi-parameter flow cytometry and imaging studies?

Leveraging USP4 antibodies for multi-parameter analyses requires careful consideration of several factors:

• Antibody conjugation selection: USP4 antibodies are available in various conjugated forms including phycoerythrin (PE), fluorescein isothiocyanate (FITC), and multiple Alexa Fluor® conjugates . Selection should be based on:

  a) Compatibility with other fluorophores: Spectral overlap must be minimized when designing multi-parameter panels

  b) Brightness requirements: For low-abundance targets, brighter fluorophores (e.g., PE, APC) may be preferable

  c) Instrument configuration: Available lasers and detectors must be considered

• Protocol optimization for intracellular staining:

  a) Fixation and permeabilization: Different reagents (paraformaldehyde, methanol, saponin) may affect epitope recognition

  b) Blocking strategy: To minimize non-specific binding, particularly important in tissues with high background

  c) Antibody titration: Determining optimal concentrations to maximize signal-to-noise ratio

• Co-staining approaches:

  a) USP4 with cell cycle markers: To investigate correlation between USP4 expression and cell cycle phase

  b) USP4 with phospho-specific markers: To examine activation of signaling pathways in USP4-expressing cells

  c) USP4 with lineage markers: Particularly valuable in heterogeneous samples like tumor biopsies

What are the current challenges in studying USP4 deubiquitinating activity, and how can antibody-based approaches address them?

Investigating USP4's enzymatic function presents unique challenges that can be addressed with specialized antibody applications:

• Identifying specific substrates:

  a) Immunoprecipitation followed by ubiquitin Western blot: USP4 has been shown to deubiquitinate multiple targets including PDPK1, TRIM21, ADORA2A, HAS2, and RHEB

  b) Proximity-dependent biotin identification (BioID): Coupling USP4 with a biotin ligase to identify proximal proteins that may be substrates

  c) TUBE (Tandem Ubiquitin Binding Entities) pulldowns: To enrich for ubiquitinated proteins, followed by mass spectrometry to identify differentially ubiquitinated proteins in USP4-manipulated conditions

• Assessing enzymatic activity:

  a) In vitro deubiquitination assays: Using recombinant USP4 and ubiquitinated substrates

  b) FRET-based sensors: To monitor USP4 activity in real-time in live cells

  c) Chain-specific ubiquitin antibodies: To determine which ubiquitin linkages (K48, K63, etc.) USP4 preferentially cleaves

• Experimental challenges and solutions:

  a) Distinguishing direct from indirect effects: Comparison of wild-type USP4 with catalytically inactive mutants

  b) Temporal dynamics: Inducible expression systems to control timing of USP4 expression

  c) Subcellular compartmentalization: USP4 can function in different cellular locations, necessitating fractionation approaches combined with immunoprecipitation

How can USP4 antibodies contribute to understanding the role of USP4 in mRNA splicing regulation?

USP4's involvement in mRNA splicing represents a complex regulatory mechanism that can be investigated using antibody-based approaches:

• Known mechanistic insights: USP4 may regulate mRNA splicing through deubiquitination of the U4 spliceosomal protein PRPF3 . This prevents PRPF3's recognition by the U5 component PRPF8, potentially destabilizing interactions within the U4/U6.U5 snRNP .

• Experimental strategies:

  a) Co-immunoprecipitation of spliceosomal complexes: Using USP4 antibodies to pull down associated splicing factors

  b) Chromatin immunoprecipitation (ChIP): To determine if USP4 associates with chromatin at sites of co-transcriptional splicing

  c) RNA immunoprecipitation (RIP): To identify RNA species associated with USP4-containing complexes

  d) Immunofluorescence co-localization: With markers of nuclear speckles or other splicing-related structures

• Functional splicing assays:

  a) Minigene splicing reporters: To assess how USP4 manipulation affects splicing of specific exons

  b) RNA-seq following USP4 knockdown/overexpression: To identify global changes in alternative splicing patterns

  c) In vitro splicing assays: Using nuclear extracts from cells with manipulated USP4 levels

How might single-cell approaches using USP4 antibodies advance our understanding of heterogeneity in USP4 function?

Single-cell analyses represent a frontier in understanding USP4 biology:

• Single-cell Western blotting:

  a) Technical considerations: Microfluidic platforms for single-cell isolation and protein separation

  b) Advantages: Can reveal cell-to-cell variation in USP4 expression levels and post-translational modifications

  c) Limitations: Lower sensitivity compared to bulk Western blotting; requires highly specific antibodies

• Mass cytometry (CyTOF):

  a) Implementation approach: Metal-conjugated USP4 antibodies combined with markers for cell state, signaling, and phenotype

  b) Data analysis: Dimensionality reduction techniques (t-SNE, UMAP) to identify cell populations with distinct USP4 expression patterns

  c) Applications: Particularly valuable in tumor samples to correlate USP4 with markers of stemness or drug resistance

• Spatial proteomics:

  a) Multiplexed immunofluorescence: Combining USP4 antibodies with other markers to preserve spatial context

  b) Imaging mass cytometry: For higher parameter analysis with spatial resolution

  c) Significance: Can reveal microenvironmental factors influencing USP4 expression and function

What are the considerations for using USP4 antibodies in combination with CRISPR-based genetic approaches?

Integrating antibody-based detection with CRISPR technologies offers powerful research strategies:

• CRISPR knockout validation:

  a) Complete vs. partial knockout: USP4 antibodies can confirm absence of protein in knockout cells or clones

  b) Off-target effects: Comparing multiple guide RNAs targeting different regions of USP4

  c) Rescue experiments: Reintroducing wild-type or mutant USP4 followed by antibody-based detection

• CRISPR activation/inhibition:

  a) CRISPRa for overexpression: Comparing endogenous upregulation via CRISPRa with traditional overexpression

  b) CRISPRi for repression: More physiological than RNAi approaches

  c) Quantification: Using USP4 antibodies to measure the degree of upregulation or repression

• CRISPR knock-in approaches:

  a) Epitope tagging: Adding small tags to endogenous USP4 for detection with tag-specific antibodies

  b) Fluorescent protein fusions: Comparing live imaging with fixed antibody-based detection

  c) Domain mutations: Introducing specific mutations to study structure-function relationships

How can proteomics approaches be combined with USP4 antibodies to identify novel interactors and substrates?

Advanced proteomics strategies can significantly expand our understanding of USP4's interactome:

• Immunoprecipitation-mass spectrometry (IP-MS):

  a) SILAC or TMT labeling: For quantitative comparison of USP4 interactors under different conditions

  b) Crosslinking strategies: To capture transient interactions

  c) Comparative analysis: Wild-type vs. catalytically inactive USP4 to distinguish substrates from other interactors

• Ubiquitinome analysis:

  a) Di-Gly remnant antibodies: To enrich ubiquitinated peptides following USP4 manipulation

  b) UbiSite approach: For proteome-wide identification of ubiquitination sites affected by USP4

  c) Integration with transcriptomics: To distinguish primary from secondary effects

• Proximity labeling proteomics:

  a) BioID or TurboID fusion with USP4: For proximity-dependent biotinylation of nearby proteins

  b) APEX2 approaches: For temporal control of labeling

  c) Subcellular targeting: Directing USP4 fusion proteins to specific compartments to identify location-specific interactions