UBE2S Antibody

What is UBE2S and what are its primary cellular functions?

UBE2S is a ubiquitin-conjugating enzyme that plays a crucial role in the ubiquitin-proteasome system with a molecular weight of approximately 22-26 kDa . It functions primarily as an E2 enzyme that accepts ubiquitin from E1 complexes and catalyzes its covalent attachment to target proteins . UBE2S specifically catalyzes 'Lys-11'-linked polyubiquitination and serves as an essential component of the anaphase promoting complex/cyclosome (APC/C), a cell cycle-regulated ubiquitin ligase that controls progression through mitosis via sequential targeting of cell cycle regulators for proteasomal degradation .

UBE2S operates in a two-step mechanism with other E2 enzymes:

• Initial priming: UBE2C/UBCH10 attaches short Lys11-linked chains to APC/C substrates

• Chain elongation: UBE2S specifically extends these chains into longer Lys11-linked ubiquitin chains on APC/C-bound substrates, enhancing their degradation by the proteasome

This activity is particularly critical for mitotic exit after prolonged spindle assembly checkpoint (SAC) activation .

For optimal experimental results with UBE2S antibodies, the following application-specific dilutions are recommended:

Always validate these dilutions in your specific experimental system, as sensitivity may vary between tissue or cell types and between different antibody lots. For recombinant antibodies, lot-to-lot consistency is typically higher, simplifying optimization protocols .

How does UBE2S contribute to mitotic progression, and how can this be experimentally demonstrated?

UBE2S plays a critical role in regulating mitotic progression through its interaction with the APC/C. To experimentally demonstrate this function, researchers can employ the following approaches:

• Mitotic substrate tracking: In control cells, APC/C substrates like Cyclin B1 and Securin are degraded within 3 hours post-release from mitotic arrest. In UBE2S-depleted cells, these substrates remain undegraded even 9 hours post-release, demonstrating UBE2S's essential role in their degradation .

• Early mitotic substrate analysis: UBE2S depletion leads to accumulation of early mitotic APC/C substrates like Cyclin A and Nek2A, which are normally degraded during pro-metaphase when the SAC is active .

• SAC bypass experiments: When the SAC is artificially inactivated (via BUBR1 depletion or Aurora-B inhibition), the requirement for UBE2S can be bypassed, indicating that UBE2S specifically helps overcome SAC-mediated inhibition of the APC/C .

These experimental approaches reveal that UBE2S acts as a rate-limiting factor for APC/C-mediated substrate degradation, particularly during recovery from prolonged SAC activation.

What is the relationship between UBE2S and the spindle assembly checkpoint (SAC)?

UBE2S has a nuanced relationship with the spindle assembly checkpoint that has significant implications for understanding mitotic regulation:

• SAC activation inhibits APC/C activity until all chromosomes are properly bi-oriented on the mitotic spindle.

• UBE2S becomes particularly important for efficient substrate degradation when APC/C activity has been compromised by prolonged SAC arrest .

• Upon SAC silencing, UBE2S enhances the formation of elongated ubiquitin chains on APC/C substrates, shifting the equilibrium between ubiquitination and deubiquitination to favor substrate degradation .

• UBE2S-depleted cells show a specific defect in silencing the SAC after drug-induced arrest, failing to degrade crucial APC/C substrates .

• This relationship appears to be general and essential across multiple cell types exposed to different anti-mitotic drugs .

The experimental evidence suggests that UBE2S functions as an unrecognized regulator of mitosis, specifically by promoting APC/C-targeted substrate degradation through ubiquitin chain elongation.

How is UBE2S expression altered in cancer, and what is its prognostic significance?

UBE2S expression shows consistent alterations across multiple cancer types with important implications for prognosis:

• Expression patterns: UBE2S is overexpressed in multiple cancer types including breast cancer and lung adenocarcinoma . In breast cancer, UBE2S overexpression correlates with higher grade, stage, and poor survival outcomes .

• Hormone receptor status correlation: In breast cancer, hormone receptor-positive (HR+) tumors demonstrate lower UBE2S expression compared to HR-negative tumors, with corresponding better survival outcomes .

• Prognostic value: Increased UBE2S expression predicts poor prognosis in both breast cancer (including ER+ breast cancer) and lung adenocarcinoma patients .

• Biomarker potential: Research suggests that UBE2S, particularly in combination with other markers like Numb (which is downregulated in cancer), could serve as novel biomarkers for cancer prognosis .

These findings establish UBE2S as a significant prognostic indicator across multiple cancer types, with particularly strong evidence in breast and lung cancers.

What molecular mechanisms underlie UBE2S-mediated cancer progression?

Research has identified several mechanisms through which UBE2S may promote cancer development and progression:

• Cell proliferation and survival: In lung adenocarcinoma, UBE2S silencing leads to reduced cell proliferation, decreased colony formation, and enhanced apoptosis. Conversely, UBE2S overexpression produces opposite effects, promoting cancer cell growth and survival .

• Tumor suppressor regulation: In breast cancer, UBE2S downregulates Numb, a potential tumor suppressor. UBE2S overexpression decreases Numb levels and enhances malignant characteristics, while UBE2S knockdown reverses these effects .

• Hypoxia signaling: UBE2S is involved in the ubiquitination and degradation of VHL, resulting in HIF1A accumulation . This represents a critical pathway in cancer progression as hypoxic signaling promotes angiogenesis and metabolic adaptations favorable to tumor growth.

• Cell cycle dysregulation: Given UBE2S's essential role in APC/C function and mitotic progression, its overexpression may contribute to genomic instability and aberrant cell division, hallmarks of cancer development .

These mechanisms provide insights into how UBE2S overexpression contributes to cancer development through multiple cellular pathways.

What are the optimal experimental designs for studying UBE2S function in cell cycle regulation?

To effectively investigate UBE2S functions in cell cycle regulation, researchers should consider the following experimental designs:

• Synchronization-release experiments:

  • Synchronize cells in mitosis using drugs like nocodazole or taxol

  • Collect mitotic cells by shake-off

  • Release cells from arrest and collect samples at defined timepoints (e.g., 3 and 9 hours post-release)

  • Analyze APC/C substrate degradation via Western blotting

• UBE2S depletion strategies:

  • Utilize siRNA or shRNA approaches for transient or stable knockdown

  • Implement CRISPR/Cas9 for complete knockout studies

  • Validate knockdown/knockout at both protein and mRNA levels

  • Assess cellular phenotypes, particularly focusing on mitotic progression

• Substrate degradation kinetics:

  • Monitor degradation of key APC/C substrates (Cyclin B1, Securin, Cyclin A, Nek2A)

  • Compare degradation rates between control and UBE2S-depleted cells

  • Correlate substrate levels with cell cycle progression markers

• SAC bypass experiments:

  • Combine UBE2S depletion with SAC inactivation (via BUBR1 depletion or Aurora-B inhibition)

  • Assess whether SAC bypass rescues the mitotic defects caused by UBE2S depletion

These approaches provide comprehensive insights into UBE2S function in cell cycle regulation while controlling for potential confounding factors.

What methodological considerations are important when using UBE2S antibodies for cancer research?

When employing UBE2S antibodies in cancer research, several methodological considerations are critical:

• Sample preparation:

  • For clinical samples: Ensure proper tissue preservation to maintain protein integrity

  • For cell lines: Consider cell cycle synchronization, as UBE2S functions vary throughout the cell cycle

  • Include appropriate positive controls (cancer cell lines with known UBE2S expression)

• Expression analysis approaches:

  • Western blotting: Validate antibody specificity; control for total protein loading

  • Immunohistochemistry: Include positive and negative controls; standardize scoring criteria

  • qRT-PCR: Design specific primers; validate with multiple housekeeping genes

• Correlation with clinical parameters:

  • Stratify samples based on pathological features (grade, stage, hormone receptor status)

  • Use appropriate statistical analyses for prognostic correlations

  • Consider multivariate analyses to account for confounding factors

• Functional validation:

  • Combine expression studies with functional assays (proliferation, colony formation, apoptosis)

  • Use multiple cell lines to ensure generalizability of findings

  • Include rescue experiments to confirm specificity of UBE2S-related phenotypes

These methodological considerations ensure robust and reproducible findings when investigating UBE2S in cancer contexts.

What are common technical challenges when working with UBE2S antibodies and how can they be resolved?

Researchers may encounter several technical challenges when working with UBE2S antibodies:

• Detection sensitivity issues:

  • Problem: Weak signal in Western blotting

  • Solutions: Optimize antibody concentration (start with 1:1000 dilution); increase protein loading; extend primary antibody incubation time; use enhanced chemiluminescence detection systems

• Specificity concerns:

  • Problem: Non-specific bands or background

  • Solutions: Validate with positive controls; perform peptide competition assays; consider monoclonal antibodies for higher specificity; optimize blocking conditions

• Species cross-reactivity limitations:

  • Problem: Antibody fails to detect UBE2S in specific species

  • Solutions: Verify sequence homology between species; select antibodies validated for your species of interest; consider using recombinant antibodies with defined epitope recognition

• Immunoprecipitation efficiency:

  • Problem: Poor IP results with UBE2S antibodies

  • Solutions: Increase antibody concentration (1:100 dilution recommended); optimize lysis conditions to preserve protein conformation; extend incubation time; consider crosslinking antibodies to beads

Addressing these challenges systematically will improve experimental outcomes and data quality in UBE2S research.

How can UBE2S research be combined with advanced techniques for mechanistic studies?

To gain deeper mechanistic insights into UBE2S function, researchers can combine basic UBE2S studies with these advanced techniques:

• Proximity-based protein interaction studies:

  • BioID or TurboID to identify proteins in close proximity to UBE2S in living cells

  • FRET or BRET to study dynamic interactions between UBE2S and potential binding partners

  • Proximity ligation assays to visualize protein-protein interactions in situ

• Ubiquitination profiling approaches:

  • Mass spectrometry to identify UBE2S-dependent ubiquitination sites

  • Ubiquitin remnant profiling to map global changes in ubiquitination patterns

  • In vitro reconstitution assays with purified components to dissect UBE2S enzymatic activity

• Live-cell imaging of UBE2S dynamics:

  • Fluorescently-tagged UBE2S to track localization throughout the cell cycle

  • FRAP (Fluorescence Recovery After Photobleaching) to measure UBE2S mobility

  • Correlative light and electron microscopy to precisely localize UBE2S at ultrastructural levels

• Systems biology approaches:

  • Integration of proteomics, transcriptomics, and functional data

  • Mathematical modeling of UBE2S-dependent ubiquitination kinetics

  • Network analysis to position UBE2S within broader cellular pathways

These advanced approaches can provide mechanistic insights that traditional biochemical methods alone cannot reveal.