OFP14 Antibody

What is USP14 and what are its primary functions in cellular pathways?

USP14 (Ubiquitin carboxyl-terminal hydrolase 14) is a deubiquitinating enzyme (DUB) that plays a critical role in the ubiquitin-proteasome system (UPS). It functions primarily as a proteasome-associated deubiquitinase which releases ubiquitin from proteasome-targeted ubiquitinated proteins . This enzyme ensures the regeneration of ubiquitin at the proteasome, acting as a reversibly associated subunit of the proteasome with a significant fraction existing in a proteasome-free state within cells .

USP14 serves several important cellular functions:

• Regeneration of ubiquitin molecules at the proteasome

• Regulation of protein degradation via the UPS

• Critical involvement in the degradation of specific proteins like the chemokine receptor CXCR4

• Physiological inhibition of endoplasmic reticulum-associated degradation (ERAD) under non-stressed conditions

Recent research has demonstrated that USP14 depletion impedes cellular proliferation, induces cell cycle arrest, and leads to a senescence-like phenotype, indicating its importance in cell cycle regulation .

What types of USP14 antibodies are available for research use?

USP14 antibodies are available in several formats optimized for different research applications:

Each antibody type offers distinct advantages depending on the research question. Polyclonal antibodies like the N-terminal USP14 antibody provide strong signal amplification through recognition of multiple epitopes, while phospho-specific antibodies enable precise detection of post-translational modifications critical for understanding USP14 activation states .

What are the recommended storage conditions and handling practices for USP14 antibodies?

For optimal performance and longevity of USP14 antibodies, researchers should follow these evidence-based storage and handling protocols:

• Short-term storage (up to 2 weeks): Refrigerate at 2-8°C

• Long-term storage: Store at -20°C in small aliquots to prevent freeze-thaw cycles

• Formulation: Typically supplied in PBS with 0.09% (W/V) sodium azide for stability

• Aliquoting: Divide into single-use portions upon first thawing to minimize freeze-thaw cycles

• Handling: Allow antibodies to reach room temperature before opening tubes to prevent condensation

• Contamination prevention: Use sterile technique when handling antibody solutions

Repeated freeze-thaw cycles can significantly diminish antibody performance through protein denaturation and aggregation. Creating single-use aliquots immediately after receiving the antibody is strongly recommended to maintain consistent experimental results.

How does phosphorylation affect USP14 function and how can antibodies detect these modifications?

USP14 phosphorylation represents a critical regulatory mechanism that directly impacts its deubiquitinating activity. Research has established that Akt phosphorylates USP14 at serine 432 (S432), which significantly enhances its catalytic activity .

Mechanistic insights into USP14 phosphorylation:

• Bacterially expressed USP14 exhibits minimal catalytic activity, but phosphorylation by Akt dramatically enhances its deubiquitinating function

• The phosphorylation can be detected using:

  • Phospho-serine-specific antibodies that recognize general serine phosphorylation

  • Phospho-S432-specific antibodies that specifically detect the Akt-mediated modification site

  • Akt phosphorylation-consensus motif (RxxS/T) antibodies

In experimental settings, researchers can detect USP14 phosphorylation through several approaches:

• Western blotting with phospho-specific antibodies after immunoprecipitation

• Phos-tag gel electrophoresis, which causes differential migration of phosphorylated proteins

• In vitro kinase assays with purified USP14 and active Akt

Critically, the phosphorylation status of USP14 corresponds directly to its enzymatic activity, which can be measured through Ub-AMC (ubiquitin-7-amido-4-methylcoumarin) hydrolysis assays. USP14 immunoprecipitated from cells coexpressing activated Akt shows significantly higher activity in Ub-AMC assays than USP14 expressed alone .

What experimental controls should be incorporated when working with USP14 antibodies?

Proper experimental controls are essential for generating reliable, reproducible, and interpretable results when working with USP14 antibodies:

Essential Positive Controls:

• Cell lines with known USP14 expression (e.g., HEK293T, H4 cells)

• Recombinant USP14 protein for antibody validation

• For phosphorylation studies: cells treated with Akt activators (e.g., insulin, growth factors)

Critical Negative Controls:

• USP14 knockout cells or USP14-depleted samples (siRNA/shRNA)

• For phospho-specific detection: samples treated with lambda phosphatase to remove phosphorylation

• For Akt-mediated phosphorylation studies: samples treated with Akt inhibitors (e.g., MK2206, AZD5363)

• Secondary antibody-only controls to assess non-specific binding

Experimental Validation Controls:

• Mutation controls: USP14 S432A mutant for phosphorylation studies

• Specificity controls: competitive blocking with immunizing peptide

• Cross-reactivity assessment with related DUBs

For phosphorylation studies specifically, researchers should include treatment controls with Akt inhibitors like MK2206 or serum deprivation conditions known to inactivate endogenous Akt, which demonstrably decrease USP14 phosphorylation levels .

How can USP14 antibodies be used to investigate the relationship between USP14 and the ubiquitin-proteasome system?

USP14 antibodies are instrumental in elucidating the complex relationship between USP14 and the ubiquitin-proteasome system through several methodological approaches:

Co-immunoprecipitation Studies:

• USP14 antibodies can precipitate proteasome complexes to study USP14's association with the proteasome

• Western blotting of immunoprecipitates with antibodies against proteasome subunits confirms association

• Quantification of proteasome-bound versus free USP14 under various cellular conditions

Functional Analysis:

• Following USP14 knockout or knockdown, monitoring ubiquitin pool changes using ubiquitin antibodies

• Measuring proteasome activity in the presence or absence of USP14 through fluorogenic peptide substrates

• Investigating the effect of USP14 phosphorylation on proteasome function

Cellular Response Studies:
Research shows that USP14 loss leads to upregulation of inducible polyubiquitin genes UBB and UBC as a compensatory mechanism to replenish decreased ubiquitin pools . This can be monitored by:

• RNA sequencing to detect transcriptional changes in ubiquitin and UPS components

• qPCR validation of specific UPS-related genes

• Western blotting with USP14 and ubiquitin antibodies to correlate protein levels

Substrate Degradation Analysis:
USP14 knockout cells show stabilization of specific proteins like p21, suggesting altered degradation kinetics . Researchers can:

• Perform cycloheximide chase experiments with USP14 antibodies to track protein degradation rates

• Compare ubiquitination status of putative substrates between wild-type and USP14-deficient cells

• Assess the impact of USP14 phosphorylation on substrate processing

How can researchers utilize USP14 antibodies to investigate its role in cell cycle regulation and senescence?

Recent findings demonstrate that USP14 depletion profoundly impacts cellular proliferation and induces a senescence-like phenotype . Researchers can employ USP14 antibodies to explore these biological processes through these methodological approaches:

Cell Cycle Analysis Protocol:

• Generate USP14 knockout or knockdown cells using CRISPR-Cas9 or siRNA approaches

• Validate USP14 depletion via western blotting with USP14 antibodies

• Perform flow cytometry with propidium iodide staining to quantify cell cycle distribution

• Use BrdU incorporation assays to measure S-phase entry

• Conduct immunofluorescence with USP14 and cell cycle marker antibodies (cyclins, CDKs)

Senescence Investigation Methodology:

• Assess senescence-associated β-galactosidase activity in USP14-depleted cells

• Examine morphological changes characteristic of senescence

• Use immunoblotting with USP14 antibodies alongside markers of senescence:

  • p21 expression (significantly increased in USP14 KO cells)

  • p16 expression

  • SASP (Senescence-Associated Secretory Phenotype) factors

Mechanistic Studies:

• Investigate p21 stabilization in USP14 KO cells through cycloheximide chase experiments

• Examine whether USP14's deubiquitinating activity directly affects p21 ubiquitination

• Perform rescue experiments by reintroducing wild-type USP14 or catalytically inactive mutants

Transcriptomic Analysis:
USP14 loss has been shown to alter the expression of numerous genes. Researchers can:

• Perform RNA sequencing on USP14 KO versus WT cells

• Validate key differentially expressed genes through qPCR

• Categorize affected pathways through Gene Ontology and pathway enrichment analysis

What techniques are optimal for studying the phosphorylation of USP14 by Akt and its functional consequences?

Studying USP14 phosphorylation by Akt requires a multifaceted approach combining biochemical, cell biological, and functional assays:

In Vitro Phosphorylation Analysis:

• Express and purify recombinant USP14 (wild-type and S432A mutant) from bacterial systems

• Perform in vitro kinase assays with active Akt and ATP

• Detect phosphorylation through:

  • Western blotting with phospho-serine antibodies

  • Phospho-S432-specific antibodies

  • Phos-tag gel electrophoresis for mobility shift detection

Cellular Phosphorylation Detection:

• Transfect cells with wild-type USP14 or S432A mutant constructs

• Modulate Akt activity through:

  • Expression of constitutively active Akt (Myr-Akt)

  • Treatment with Akt inhibitors (MK2206, AZD5363)

  • Serum deprivation to inactivate endogenous Akt

• Immunoprecipitate USP14 and detect phosphorylation with phospho-specific antibodies

Functional Impact Assessment:

Physiological Significance Investigation:

• Generate cell lines expressing phospho-mimetic (S432D/E) or phospho-deficient (S432A) USP14 mutants

• Analyze cellular phenotypes including proliferation, cell cycle progression, and response to proteasome inhibitors

• Perform proteomics to identify differentially regulated substrates

What methodological approaches can be used to troubleshoot inconsistent results when using USP14 antibodies?

When researchers encounter inconsistent results with USP14 antibodies, a systematic troubleshooting approach is essential:

Antibody Validation Protocol:

• Confirm antibody specificity using:

  • USP14 knockout or knockdown cells as negative controls

  • Western blotting against recombinant USP14 protein

  • Peptide competition assays with the immunizing peptide

Sample Preparation Optimization:

• Test multiple lysis buffers to ensure efficient extraction of USP14:

  • RIPA buffer for most applications

  • NP-40 or Triton X-100 based buffers for preserving protein interactions

  • SDS-based buffers for difficult-to-extract fractions

• Add protease and phosphatase inhibitors to prevent degradation and dephosphorylation

• For phosphorylation studies, lyse cells directly in SDS sample buffer to preserve modifications

Technical Parameter Adjustment:

• Optimize antibody dilutions using a dilution series (start with manufacturer recommendations)

• Adjust incubation times and temperatures

• For Western blotting:

  • Test different blocking agents (BSA vs. milk)

  • Vary transfer conditions for different molecular weight proteins

  • Consider wet transfer for better efficiency with larger proteins

Cross-Reactivity Assessment:

• Test the antibody against a panel of related DUBs to check for cross-reactivity

• For polyclonal antibodies, consider affinity purification against the immunizing peptide

• Compare results using antibodies targeting different epitopes of USP14

Application-Specific Considerations:

• For immunoprecipitation: Optimize antibody-to-beads ratio and washing stringency

• For immunohistochemistry: Test multiple antigen retrieval methods and fixation protocols

• For immunofluorescence: Compare different fixation methods (paraformaldehyde vs. methanol)

How can USP14 antibodies be utilized in studying cancer pathways and potential therapeutic applications?

USP14 has emerged as a significant player in cancer biology through its role in protein homeostasis and cell cycle regulation. Researchers can employ USP14 antibodies to explore cancer-related pathways through these approaches:

Analysis of USP14 Expression in Cancer Models:

• Compare USP14 protein levels across cancer cell lines and matched normal tissues using western blotting

• Perform immunohistochemistry on tissue microarrays to evaluate USP14 expression in patient samples

• Correlate USP14 expression with clinical parameters and patient outcomes

Investigation of USP14-Akt Axis in Cancer:

• Examine the correlation between Akt activation and USP14 phosphorylation in cancer cells

• Analyze the effect of Akt inhibitors on USP14 activity in cancer models

• Investigate whether USP14 phosphorylation status can serve as a biomarker for Akt activation in tumors

Therapeutic Target Exploration:

• Use USP14 antibodies to monitor protein levels and activity following treatment with:

  • Proteasome inhibitors (bortezomib, carfilzomib)

  • USP14-specific small molecule inhibitors

  • Akt pathway modulators

• Perform combination treatment studies to identify synergistic therapeutic approaches

• Develop sandwich ELISA or proximity ligation assays to detect USP14-proteasome association as a readout for inhibitor efficacy

Pathway Analysis:
The loss of USP14 has been shown to affect transcriptome profiles, including cell cycle regulators . Researchers can:

• Perform pathway analysis of differentially expressed genes in USP14-depleted cancer cells

• Validate key nodes in identified pathways through protein expression analysis

• Investigate whether USP14 inhibition can synergize with other cancer therapeutics targeting identified pathways

What are the optimal protocols for using USP14 antibodies in different experimental applications?

Western Blotting Protocol:

• Sample preparation: Lyse cells in RIPA buffer with protease/phosphatase inhibitors

• Protein quantification: Use BCA or Bradford assay to normalize loading

• SDS-PAGE: Load 20-50 μg of protein per lane

• Transfer: Use PVDF membrane for optimal protein binding

• Blocking: 5% non-fat milk or BSA in TBST for 1 hour at room temperature

• Primary antibody: Dilute USP14 antibody 1:1000 in blocking buffer, incubate overnight at 4°C

• Washing: 3 × 10 minutes with TBST

• Secondary antibody: Anti-rabbit HRP conjugate at 1:5000, 1 hour at room temperature

• Detection: Use enhanced chemiluminescence (ECL) substrate

Immunohistochemistry Protocol:

• Sample preparation: Formalin-fixed, paraffin-embedded sections (4-5 μm)

• Deparaffinization: Xylene and graded ethanol series

• Antigen retrieval: Citrate buffer (pH 6.0), 95°C for 20 minutes

• Blocking: 3% hydrogen peroxide (10 min) followed by 5% normal goat serum (1 hour)

• Primary antibody: USP14 antibody at 1:50-1:100 dilution, overnight at 4°C

• Detection: HRP-polymer detection system with DAB substrate

• Counterstain: Hematoxylin

• Dehydration and mounting: Ethanol series, xylene, permanent mounting medium

Immunoprecipitation for Phosphorylation Studies:

• Cell lysis: Use NP-40 buffer with phosphatase inhibitors

• Pre-clearing: Incubate lysate with protein A/G beads for 1 hour

• Immunoprecipitation: Add USP14 antibody (2-5 μg) to 500 μg lysate, rotate overnight at 4°C

• Bead binding: Add protein A/G beads, incubate 2-4 hours

• Washing: 4 × with lysis buffer containing phosphatase inhibitors

• Elution: SDS sample buffer at 95°C for 5 minutes

• Analysis: Western blotting with phospho-specific antibodies

How can researchers design experiments to investigate the relationship between USP14 phosphorylation and its biological functions?

To comprehensively investigate how phosphorylation affects USP14 function, researchers should implement this experimental design framework:

Generation of Phosphorylation-Site Mutants:

• Create expression constructs for:

  • Wild-type USP14

  • Phospho-deficient mutant (S432A)

  • Phospho-mimetic mutant (S432D or S432E)

• Validate expression by western blotting with USP14 antibodies

• Confirm phosphorylation status using phospho-specific antibodies

Enzymatic Activity Assessment:

• Perform Ub-AMC hydrolysis assays to compare DUB activity:

  • Purify recombinant proteins from bacterial or mammalian expression systems

  • Measure fluorescence release kinetics in the presence of Ub-AMC substrate

  • Compare activity before and after in vitro phosphorylation by Akt

• Analyze global ubiquitination patterns in cells expressing different USP14 variants

Cellular Localization Studies:

• Perform subcellular fractionation and immunoblotting to determine if phosphorylation affects USP14 distribution

• Use immunofluorescence microscopy to visualize USP14 localization in cells expressing different USP14 variants

• Examine co-localization with proteasome subunits to assess if phosphorylation affects proteasome association

Functional Impact Analysis:

• Establish stable cell lines expressing wild-type or mutant USP14 in a USP14-depleted background

• Assess cellular phenotypes:

  • Proliferation rates and cell cycle distribution

  • Sensitivity to proteasome inhibitors

  • Ability to degrade specific proteasome substrates

• Perform cycloheximide chase experiments to measure protein half-lives

• Examine the stabilization of known USP14-regulated proteins like p21

Signaling Pathway Integration:

• Modulate Akt activity through growth factors, inhibitors, or constitutively active/dominant negative mutants

• Monitor USP14 phosphorylation and activity in response to various cellular stresses

• Investigate cross-talk with other post-translational modifications of USP14

What are the key considerations for developing and validating new phospho-specific USP14 antibodies?

Development and validation of phospho-specific USP14 antibodies requires rigorous methodology to ensure specificity and reliability:

Antigen Design Considerations:

• Synthesize phosphopeptides corresponding to the Ser432 region of USP14

• Include several amino acids on either side of the phosphorylation site for context

• Prepare both phosphorylated and non-phosphorylated peptides for screening

• Consider carrier protein conjugation (KLH, BSA) for immunization

Immunization and Antibody Production:

• Immunize rabbits or other suitable host animals with the phosphopeptide-carrier conjugate

• Collect antisera and monitor antibody titers by ELISA

• For monoclonal antibodies, perform hybridoma screening with both phosphorylated and non-phosphorylated peptides

Purification Strategies:

• Perform negative selection using non-phosphorylated peptide columns to remove antibodies recognizing the backbone

• Use positive selection with phosphopeptide affinity columns to isolate phospho-specific antibodies

• Test eluted fractions by ELISA against both peptide forms

Validation Protocol:

• Western blotting with:

  • Recombinant USP14 with and without Akt-mediated phosphorylation

  • Wild-type USP14 versus S432A mutant expressed in cells

  • Cell lysates before and after phosphatase treatment

• Immunoprecipitation tests:

  • Precipitate USP14 from cells with active or inhibited Akt

  • Probe with general USP14 antibodies to confirm protein capture

• Specificity controls:

  • Peptide competition assays with phosphorylated and non-phosphorylated peptides

  • Cross-reactivity testing against related phosphorylation sites

Application-Specific Validation:

• For immunohistochemistry: Compare staining patterns in tissues with known Akt activation status

• For immunofluorescence: Co-staining with total USP14 antibodies and verification with S432A mutant

• For ELISA development: Establish detection limits and dynamic range