USP48 Antibody

What are the primary applications of USP48 antibodies in research?

USP48 antibodies are versatile research tools applicable across multiple experimental techniques. The most common applications include:

• Western Blotting (WB): The predominant application, with typical working dilutions ranging from 1:500 to 1:2000. USP48 appears at approximately 119 kDa on immunoblots .

• Immunoprecipitation (IP): Effective for studying protein-protein interactions involving USP48, such as its associations with TRAF2, Aurora B, and RELA .

• Immunofluorescence (IF): Useful for determining subcellular localization, particularly for examining USP48 recruitment to DNA damage sites and its co-localization with targets .

• Immunohistochemistry (IHC): Valuable for tissue expression studies, especially when examining USP48 levels in tumor samples versus normal tissues .

• ELISA: Provides quantitative measurements of USP48 protein levels .

For optimal results, each technique requires specific antibody concentrations, sample preparation protocols, and detection methods tailored to the experimental question.

How do USP48 antibodies differ in their epitope recognition and applications?

USP48 antibodies target different regions of the protein, affecting their utility in specific applications:

When selecting an antibody, researchers should consider:

• The specific domain of interest (DUSP domains, ubiquitin-like domain, catalytic site)

• Whether post-translational modifications might affect epitope recognition

• The presence of alternative splicing variants (USP48 has seven identified isoforms)

• Whether denatured or native protein detection is required

What controls should be included when validating a USP48 antibody?

Proper experimental controls are essential for antibody validation:

• Positive controls: Cells or tissues known to express USP48 (widely expressed, but higher in glioblastoma than in grade II astrocytoma) .

• Negative controls:

  • Primary antibody omission

  • Non-specific IgG of the same isotype and host species

  • USP48 knockdown/knockout samples using siRNA or CRISPR/Cas9

• Specificity controls:

  • Comparing USP48 detection against other USP family members (e.g., USP11 as demonstrated in studies)

  • Pre-adsorption with immunogenic peptide

  • Testing multiple antibodies targeting different epitopes

A comprehensive validation should include:

• Molecular weight verification (119 kDa for full-length USP48)

• Signal reduction upon USP48 depletion

• Consistent localization patterns across different detection methods

• Cross-reactivity assessment with related deubiquitinases

How can USP48 antibodies be used to investigate its role in protein stabilization pathways?

USP48 has been shown to stabilize several proteins through deubiquitination, including TRAF2, Gli1, Aurora B, and p65/RELA. To study these mechanisms:

• Co-immunoprecipitation assays:

  • Use USP48 antibodies to pull down potential substrates or vice versa

  • Include controls using catalytically inactive USP48 mutants (C98S)

  • Confirm interactions with reciprocal IPs

• Protein stability assays:

  • Combine cycloheximide (CHX) chase with USP48 knockdown/overexpression

  • Quantify target protein half-life using time-course immunoblots

  • Compare wild-type USP48 effects to catalytically inactive mutants

• Ubiquitination analysis:

  • Perform in vivo ubiquitination assays with HA/His-tagged ubiquitin

  • Use proteasome inhibitors (MG132) to accumulate ubiquitinated species

  • Immunoprecipitate the substrate protein and blot for ubiquitin

  • Compare ubiquitination levels in the presence/absence of USP48

• In vitro deubiquitination assays:

  • Purify recombinant USP48 and ubiquitinated substrate

  • Incubate and monitor ubiquitin removal using anti-ubiquitin antibodies

  • Include controls with catalytically inactive USP48

For example, studies demonstrated that USP48 stabilizes TRAF2 by deubiquitination, with USP48 knockdown reducing TRAF2 protein levels without affecting mRNA expression .

What are the methodological approaches for studying USP48's role in cell cycle regulation?

USP48 plays a critical role in cell cycle progression, particularly through its regulation of Aurora B. Researchers can investigate this function using:

• Cell cycle synchronization and analysis:

  • Synchronize cells using thymidine block or nocodazole

  • Track USP48 and Aurora B levels across cell cycle phases

  • Use flow cytometry to assess cell cycle distribution after USP48 manipulation

• Mitotic phenotype characterization:

  • Immunofluorescence microscopy to visualize mitotic structures

  • Quantify mitotic defects (lagging chromosomes, misaligned chromosomes, multipolar spindles)

  • Track cytokinesis progression using time-lapse imaging

• Mitotic protein stability assessment:

  • Monitor Aurora B levels throughout mitosis with and without USP48

  • Determine if USP48 depletion accelerates Aurora B degradation

  • Assess impacts on downstream targets of Aurora B

• Chromosome segregation analysis:

  • Use fluorescently labeled histones to track chromosome movement

  • Quantify segregation errors in USP48-depleted cells

  • Correlate with Aurora B activity markers

Research has demonstrated that USP48 depletion results in:

• Reduced Aurora B protein levels

• Increased mitotic defects (lagging chromosomes: 28.6% vs 8.5% in control)

• Delayed progression through the cell cycle

• Cytokinesis failure leading to multinucleated cells

How can I design experiments to study USP48's involvement in the DNA damage response?

USP48 has been implicated in the DNA damage response through its interaction with BRCA1. Appropriate experimental approaches include:

• DNA damage induction and recruitment studies:

  • Induce localized DNA damage using laser micro-irradiation

  • Track USP48 recruitment to damage sites using immunofluorescence

  • Determine co-localization with damage response proteins (53BP1, BRCA1)

• DNA repair pathway analysis:

  • Measure homologous recombination efficiency using reporter assays

  • Assess single-strand annealing (SSA) in USP48-depleted cells

  • Quantify RAD51 foci formation after DNA damage

• H2A ubiquitination studies:

  • Examine BRCA1-mediated H2A ubiquitination with/without USP48

  • Use site-specific ubiquitin antibodies to detect H2A modification

  • Perform in vitro deubiquitination assays with nucleosome substrates

• Drug sensitivity assays:

  • Test sensitivity to DNA damaging agents (e.g., camptothecin)

  • Measure cell survival and apoptosis rates

  • Determine RAD52 dependency of observed effects

Research has shown that USP48 depletion:

• Increases DNA end resection and RAD51 recruitment

• Enhances single-strand annealing (SSA)

• Confers RAD52-dependent survival advantage to camptothecin-treated cells

What approaches can be used to evaluate USP48's function in the NF-κB signaling pathway?

USP48 regulates NF-κB signaling through interactions with RELA/p65 and TRAF2. To investigate this role:

• NF-κB activation assays:

  • Use luciferase reporter constructs containing NF-κB response elements

  • Stimulate cells with TNFα and measure reporter activity

  • Compare responses with USP48 knockdown/overexpression

• p65 nuclear translocation and stability:

  • Perform cellular fractionation to separate nuclear and cytosolic components

  • Immunoblot for p65 in each fraction after TNFα stimulation

  • Quantify p65 degradation rates with cycloheximide chase

• TRAF2 stabilization analysis:

  • Compare TRAF2 levels after USP48 manipulation

  • Assess TRAF2 ubiquitination status through IP-Western

  • Determine effects on downstream signaling components

• Target gene expression analysis:

  • Measure NF-κB target gene expression using qRT-PCR

  • Perform ChIP assays to assess p65 recruitment to promoters

  • Compare the expression profiles with USP48 modulation

Research findings demonstrate that:

• USP48 knockdown stabilizes p65, particularly in the nuclear compartment

• p65 levels significantly increased in USP48-silenced cells

• USP48 silencing enhances NF-κB transcriptional activity

• USP48 depletion reduces TRAF2 protein levels without affecting mRNA expression

How can I investigate USP48's role in glioblastoma progression?

USP48 has been implicated in glioblastoma tumorigenesis through stabilization of Gli1. Researchers studying this connection should consider:

• Expression correlation studies:

  • Compare USP48 and Gli1 expression in glioma specimens of different grades

  • Use immunohistochemistry to assess protein levels in patient samples

  • Perform correlation analysis between USP48 expression and glioma malignancy

• Hedgehog pathway modulation:

  • Utilize Gli-responsive luciferase reporters to measure pathway activity

  • Compare the effects of wild-type and catalytically inactive USP48

  • Assess changes in Gli1 target gene expression (N-Myc, Cyclin D2, Bmi1)

• Glioma stem cell models:

  • Evaluate USP48's impact on neurosphere formation

  • Assess stem cell marker expression after USP48 depletion

  • Compare effects in glioma stem cells versus normal astrocytes

• In vivo tumorigenesis assays:

  • Establish orthotopic xenograft models with USP48-depleted glioma cells

  • Monitor tumor growth and survival outcomes

  • Analyze Gli1 levels and target gene expression in tumors

Research has revealed:

• Positive correlation between USP48 and Gli1 expression in glioblastoma samples (r = 0.6170, P < 0.001)

• Significantly higher USP48 expression in glioblastomas compared to grade II astrocytomas (P = 0.0006)

• USP48 knockdown inhibits neurosphere formation and stem cell proliferation

• USP48 depletion has minimal effect on normal human astrocytes

What techniques are appropriate for studying USP48's role in ciliogenesis and tubulin modification?

USP48 has been implicated in ciliary function and tubulin modification. To investigate this aspect:

• Ciliogenesis assessment:

  • Induce primary cilia formation through serum starvation

  • Immunostain for ciliary markers (acetylated α-tubulin, ARL13B)

  • Measure ciliary length and frequency in USP48-depleted cells

• Tubulin acetylation analysis:

  • Quantify acetylated α-tubulin levels by immunofluorescence

  • Compare ciliary vs. cytoplasmic tubulin acetylation

  • Perform western blotting for total vs. acetylated tubulin

• Ciliary protein stability studies:

  • Examine levels of retinal degeneration-associated proteins (ARL3, UNC119)

  • Perform cycloheximide chase to determine protein half-lives

  • Compare ubiquitination patterns with/without USP48

• Live cell imaging of ciliary transport:

  • Use fluorescently tagged ciliary cargo proteins

  • Track intraflagellar transport in real-time

  • Quantify transport rates and directionality

Research findings show that USP48 knockdown:

• Does not significantly alter ciliary length or the percentage of ciliated cells

• Significantly increases the fluorescence intensity of ciliary acetylated α-tubulin

• Modulates ciliary and synaptic transport important for photoreceptor function

How can I use USP48 antibodies to study its role in hematological malignancies?

USP48 has been implicated in acute myeloid leukemia (AML). To investigate this connection:

• Apoptotic cleavage analysis:

  • Induce apoptosis using chemotherapeutic agents

  • Detect full-length USP48 (119 kDa) and cleaved fragments

  • Identify the caspase-3 cleavage site (DEQD at positions 611-614)

• Functional studies in AML cell lines:

  • Generate USP48 knockdown models using shRNA

  • Assess proliferation, colony formation, and cell cycle distribution

  • Measure apoptotic rates after chemotherapy treatment

• Substrate identification in AML:

  • Perform immunoprecipitation with USP48 antibodies

  • Use mass spectrometry to identify binding partners

  • Validate interactions through reciprocal IP and functional assays

• Chemosensitivity testing:

  • Combine USP48 knockdown with chemotherapy agents (HHT, Ara-C, ATRA)

  • Measure cell viability and apoptosis rates

  • Determine synergistic effects through combination index analysis

Research has demonstrated that in AML:

• USP48 is cleaved by caspase-3 at the DEQD motif during drug-induced apoptosis

• The N-terminal fragment containing the catalytic domain is rapidly degraded

• USP48 knockdown reduces colony formation, induces G1 arrest, and promotes apoptosis

• Inhibition of USP48 enhances sensitivity to chemotherapy drugs (MTT assay showed combined effects of USP48 shRNA and HHT)

What methodologies can address contradictory findings in USP48 research?

Some studies show seemingly contradictory roles for USP48 in different cellular contexts. To address these contradictions:

• Cell type-specific function analysis:

  • Compare USP48 effects across multiple cell lines (epithelial vs. immune vs. neural)

  • Identify cell type-specific binding partners through immunoprecipitation

  • Determine if alternative splicing produces different isoforms in various tissues

• Context-dependent signaling studies:

  • Examine USP48 function under different stimuli (TNFα, DNA damage, cell cycle phases)

  • Compare acute vs. chronic USP48 depletion effects

  • Determine if post-translational modifications alter USP48 activity

• Substrate competition analysis:

  • Overexpress multiple USP48 substrates to identify preferential targeting

  • Use domain mapping to identify substrate-specific interaction regions

  • Determine if different substrates compete for USP48 binding

• Integrated multi-omics approach:

  • Combine proteomics, transcriptomics, and ubiquitinomics

  • Identify ubiquitination changes after USP48 manipulation

  • Correlate changes with functional outcomes in specific pathways

For example, USP48 has been shown to both promote cell survival in glioblastoma yet enhance apoptosis sensitivity in AML , likely reflecting context-dependent functions and different substrates in these cellular environments.

What are the critical considerations for optimizing USP48 detection in western blotting?

Successful western blotting for USP48 requires attention to several technical aspects:

• Sample preparation:

  • Include protease inhibitors to prevent degradation

  • Add deubiquitinase inhibitors (NEM, PR-619) to preserve ubiquitinated species

  • Use phosphatase inhibitors if studying phosphorylated forms of USP48

• Gel percentage and run conditions:

  • Use 8-10% SDS-PAGE gels for optimal separation of the 119 kDa protein

  • Consider gradient gels when examining both full-length USP48 and cleavage products

  • Allow sufficient run time for proper resolution of high molecular weight proteins

• Transfer parameters:

  • Employ wet transfer methods for large proteins

  • Consider extended transfer times (>90 minutes) or lower currents

  • Use PVDF membranes for stronger protein binding

• Antibody optimization:

  • Typical dilutions range from 1:500 to 1:2000 for western blotting

  • Include 5% BSA in blocking buffer to reduce background

  • Optimize incubation time and temperature (4°C overnight often yields best results)

• Common pitfalls and solutions:

  • High background: Increase blocking time, add Tween-20 to wash buffers

  • Weak signal: Increase antibody concentration, extend exposure time

  • Multiple bands: Verify with knockout controls, consider alternative antibodies

How should researchers design deubiquitination assays to evaluate USP48 activity?

Proper design of deubiquitination assays is crucial for studying USP48 function:

• In vitro deubiquitination assays:

  • Substrate preparation: Use recombinantly expressed and purified ubiquitinated proteins

  • Enzyme source: Immunoprecipitated USP48-V5 or recombinantly expressed protein

  • Reaction buffer: 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 1 mM ATP, 5 mM MgCl₂, 1 mM DTT

  • Controls: Include catalytically inactive USP48 mutant (C98S)

• Ub-AMC fluorescence assays:

  • Measure deubiquitinating activity using ubiquitin-7-amino-4-methylcoumarin

  • Monitor fluorescence release over time as indicator of DUB activity

  • Compare wild-type USP48 with mutants or in presence of inhibitors

• Cellular ubiquitination assays:

  • Transfect cells with HA-tagged ubiquitin and USP48

  • Treat with proteasome inhibitors before lysis

  • Immunoprecipitate substrate proteins and blot for ubiquitin

  • Compare ubiquitination patterns with/without USP48 overexpression or knockdown

• Chain-specific deubiquitination analysis:

  • Use linkage-specific ubiquitin antibodies (K48, K63, etc.)

  • Determine USP48's preference for different ubiquitin chain types

  • Correlate chain specificity with functional outcomes (degradation vs. signaling)

Research has demonstrated USP48's ability to cleave ubiquitin from various substrates including TRAF2, Gli1, Aurora B, and H2A-ubiquitin, with specificity for certain substrates and ubiquitin chain types .

What methodologies can differentiate between USP48's catalytic and scaffolding functions?

USP48 may exert both catalytic and non-catalytic functions. To distinguish between these roles:

• Catalytically inactive mutants:

  • Compare wild-type USP48 with C98S mutant (disrupts catalytic activity)

  • Assess effects on substrate stability and downstream pathway activation

  • Determine if mutant retains binding capacity despite loss of enzymatic function

• Domain deletion constructs:

  • Generate constructs lacking specific domains (DUSP, UBL, catalytic)

  • Examine interaction profiles with known binding partners

  • Assess ability to rescue phenotypes in USP48-depleted cells

• Structure-function analysis:

  • Create USP48 mutants with deleted amino acids 886-890 (affects function)

  • Compare deubiquitinating activity in vitro

  • Assess substrate binding capabilities independently of catalytic activity

• Chemical inhibition approach:

  • Use deubiquitinase inhibitors while maintaining USP48 protein presence

  • Determine which functions persist despite catalytic inhibition

  • Compare to genetic depletion which removes both catalytic and scaffolding functions

Studies have shown that:

• USP48 C98S mutant acts as a dominant-negative, decreasing endogenous TRAF2 levels

• Deletion of residues 886-890 affects USP48 function while preserving protein interaction

• USP48 requires an additional ubiquitin that itself is not cleaved for full activity on H2A-ubiquitin nucleosomes

These findings highlight the importance of distinguishing between enzymatic and structural roles of USP48 in experimental design and interpretation.