CDC20-5 Antibody

What is CDC20 and what cellular processes does it regulate?

CDC20 serves as an essential regulator in cell cycle progression, performing two critical functions: it promotes mitotic exit by activating the Anaphase-Promoting Complex/Cyclosome (APC/C) and monitors kinetochore-microtubule attachment through activation of the Spindle Assembly Checkpoint (SAC) . As a key component of the cell cycle machinery, CDC20 ensures proper chromosome segregation during mitosis by preventing premature sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle. When CDC20 is depleted through knockout or RNAi approaches, cells typically arrest at metaphase and eventually undergo apoptosis, highlighting its indispensable role in mitotic progression . The protein contains several functional domains, including the WD40 domain, which has been implicated in its transcriptional regulatory activities and protein-protein interactions .

What experimental techniques are most effective for detecting CDC20 expression?

Multiple complementary techniques should be employed for robust detection of CDC20 expression:

• Immunofluorescence Microscopy: Particularly useful for quantifying CDC20 expression during metaphase when protein levels peak. This allows single-cell analysis of CDC20 expression patterns throughout the cell cycle .

• Western Blotting: For bulk quantification of CDC20 protein levels, cells should be synchronized at prometaphase (typically using nocodazole treatment) to control for cell cycle-dependent expression fluctuations . When performing western blots, fluorescently labeled secondary antibodies and quantitative scanning (e.g., using LI-COR Odyssey CCD scanner) provide the most reliable quantitative results .

• Immunoprecipitation: For studying CDC20's interactions with binding partners such as Cdc27 and CBP. Cell lysates can be prepared using freeze-thaw cycles in appropriate buffers (50 mM Tris pH 7.5, 15 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 0.01% SDS) containing protease inhibitors .

• Mass Spectrometry: For detecting CDC20 peptides when western blot signals are below detection threshold, as demonstrated in studies detecting residual CDC20 in presumed knockout cell lines .

How does CDC20 expression vary throughout the cell cycle?

CDC20 expression exhibits significant fluctuation throughout the cell cycle, with peak expression occurring during metaphase . This dynamic expression pattern necessitates careful experimental design when studying CDC20. For accurate quantification, cells must be synchronized or analyses must be performed on specific cell cycle stages. When comparing CDC20 levels between different cell populations, synchronization with nocodazole to arrest cells at prometaphase provides a standardized time point for comparison . Additionally, multiple CDC20 protein isoforms exist that may affect mitotic duration, though their relative abundance does not appear to differ significantly between diploid and aneuploid cells .

How does the Mitotic Checkpoint Complex (MCC) interact with CDC20 to inhibit APC/C activity?

The interaction between the Mitotic Checkpoint Complex (MCC) and CDC20 involves a sophisticated molecular mechanism that ensures proper cell cycle regulation:

The MCC inhibits APC/C activity through a dual-CDC20 mechanism, where the MCC binds and inhibits a second CDC20 molecule that has already bound and activated the APC/C . The core MCC consists of MAD2, BUBR1, and CDC20 in a 1:1:1 ratio, forming what is called the "core MCC" complex . This complex can potently inhibit active APC/C-CDC20 complexes within 10 minutes of interaction.

The inhibitory mechanism involves specific domains in these proteins:

• The D-box of BUBR1 is crucial for binding a second CDC20 molecule

• CDC20's KEN-box recognition motif (KR) is essential for core MCC formation

• CDC20's D-box recognition motif (DR) is required for inhibiting a second CDC20

When these interactions are disrupted through mutations (such as ΔDR or ΔKR mutations in CDC20 or ΔD-box mutations in BUBR1), the spindle assembly checkpoint becomes defective, leading to chromosome segregation errors . These findings demonstrate that SAC functionality depends on the core MCC's ability to inhibit a second CDC20 molecule, even when it's part of an already active APC/C-CDC20 complex.

What role does CDC20 play in transcriptional regulation?

Beyond its well-established role in mitotic progression, CDC20 demonstrates transcriptional regulatory functions that impact cell cycle-related gene expression. Research has shown that CDC20 transcriptionally upregulates UbcH10 expression, with the WD40 domain of CDC20 being required for this activity . This finding reveals an additional layer of cell cycle regulation, where CDC20 not only functions as a mitotic regulator through protein-protein interactions but also influences gene expression directly.

The transcriptional activity of CDC20 likely involves interactions with transcriptional machinery components. Immunoprecipitation experiments have demonstrated interactions between CDC20, Cdc27, and CREB-binding protein (CBP), suggesting a potential mechanism for CDC20's transcriptional regulatory activities . These interactions provide insight into how CDC20 may integrate mitotic progression with gene expression regulation, ensuring coordinated cell cycle progression.

Methodologically, to study CDC20's transcriptional activity, researchers should employ chromatin immunoprecipitation (ChIP) assays to identify direct DNA binding sites, coupled with reporter gene assays to validate the functional significance of these interactions. RNA-seq or qPCR analysis following CDC20 manipulation (overexpression or depletion) can further elucidate the broader transcriptional networks influenced by CDC20 activity.

How do CDC20 expression levels correlate with sensitivity to Spindle Assembly Checkpoint inhibition?

CDC20 expression levels demonstrate a strong correlation with cellular sensitivity to SAC inhibition, particularly in the context of cancer and aneuploidy:

• Expression patterns in aneuploid cells: Highly aneuploid cancer cells significantly overexpress CDC20 mRNA compared to near-diploid cells . This overexpression can be quantified at both the mRNA level (through transcriptomic analysis) and protein level (through western blotting of synchronized cells or immunofluorescence microscopy of metaphase cells) .

• Causal relationship with drug sensitivity: Statistical analysis using linear regression models reveals that CDC20 expression levels are a major determinant of differential responses to MPS1 inhibitors and genetic disruption of SAC components . When CDC20 expression is included as a covariate in these models, the significant association between aneuploidy and response to SAC inhibition is completely abolished .

• Experimental validation: Depletion of CDC20 using siRNA or shRNA approaches reduces sensitivity to SAC inhibition, confirming the causal relationship between CDC20 levels and response to these inhibitors . This effect has been demonstrated across multiple cell systems, including:

  • Human colon cancer cell line HCT116 and its aneuploid derivatives

  • Immortalized epithelial cell line RPE1 and its aneuploid derivatives

  • Mouse cells with various transformation states

These findings suggest that high CDC20 expression could serve as a potential biomarker for identifying tumors that might respond favorably to SAC inhibition therapy .

How does CDC20 depletion affect mitotic progression and chromosomal instability?

CDC20 depletion has profound effects on mitotic progression and chromosomal stability across various cell types:

• Extended metaphase duration: Depletion of CDC20 via siRNA or shRNA significantly extends metaphase duration in both mouse and human cell lines . This extension of metaphase provides cells additional time to correct erroneous kinetochore-microtubule attachments, potentially reducing mitotic errors.

• Reduced mitotic aberrations: Cells with depleted CDC20 show significantly decreased prevalence and severity of mitotic aberrations when exposed to SAC inhibition . The effect is observed across multiple cell types, including:

  • Mouse and human 3T3 and HCT116 cells

  • Highly aneuploid HPT1 and HPT2 cell lines

  • HCT116 cells with induced aneuploidy

• Impact on chromosomal instability (CIN): CDC20 depletion significantly alleviates chromosomal instability induced by SAC inhibition . The severity of mitotic aberrations is reduced, leading to more stable genomic content over time.

• Mechanism of action: The protective effect of CDC20 depletion against SAC inhibition-induced CIN likely stems from the prolonged metaphase, which allows cells more time to establish proper kinetochore-microtubule attachments despite compromised checkpoint function .

For experimental investigation of these effects, live-cell imaging with fluorescently labeled chromosomes provides the most comprehensive data, allowing researchers to track mitotic progression in real-time and categorize mitotic aberrations according to their severity .

How should CDC20 antibodies be validated for specificity in experimental applications?

Comprehensive validation of CDC20 antibodies requires multiple approaches to ensure specificity and reliability:

What are the optimal conditions for immunoprecipitation of CDC20 and its binding partners?

Successful immunoprecipitation of CDC20 and its interaction partners requires careful consideration of buffer composition, antibody selection, and experimental conditions:

Buffer Composition:
For general CDC20 interactions:

• HEPES buffer (150 mM KCl, 20 mM HEPES pH 7.8, 10 mM EDTA, 10% Glycerol, 0.2% NP-40, 1 mM DTT) supplemented with protease inhibitors (Roche complete inhibitor cocktail tablet), phosphatase inhibitors (0.2 μM microcystin), and PMSF (1 mM)

For interactions with transcriptional regulators:

• Tris buffer (50 mM Tris pH 7.5, 15 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 0.01% SDS) containing protease inhibitor mixture

Cell Lysis Protocol:

• For standard interactions, incubate cells with lysis buffer for 10 minutes on ice, followed by clarification through centrifugation at 20,000 × g for 10 minutes

• For transcriptional complex isolation, freeze-thaw cycles provide effective lysis while preserving protein-protein interactions

Antibody Selection and Coupling:

• Anti-CDC20, anti-Cdc27, and anti-CBP antibodies have been successfully used for CDC20 complex immunoprecipitation

• For APC/C-CDC20 complex isolation, antibodies against APC3 (AF3.1) or APC4 have proven effective

• Covalently couple antibodies to Protein G Dynabeads (Invitrogen) for optimal results

Controls:

• Normal IgG should be included as a negative control for all immunoprecipitation experiments

• For interaction studies, include additional controls with antibodies against predicted interaction partners

What approaches are most effective for studying CDC20 function through gene depletion or mutation?

Multiple complementary approaches can be employed to study CDC20 function through depletion or mutation:

RNAi-based Approaches:

• siRNA: Provides rapid, transient knockdown. Most effective when cells are synchronized by double thymidine block prior to transfection

• shRNA: Offers more stable, long-term depletion for extended studies of CDC20 function

CRISPR/Cas9 Gene Editing:

• Complete knockout attempts typically result in cell death due to metaphase arrest and apoptosis

• Conditional knockout systems using inducible Cas9 or inducible degron tags provide more controlled manipulation of CDC20 levels

• Analysis of CRISPR knockout clones requires careful validation, as cells surviving apparent knockout may retain residual CDC20 not detectable by western blot but identifiable through mass spectrometry

3. Rescue Experiments with Mutant Variants:
Multiple CDC20 mutants have been characterized that can be used in rescue experiments:

• CDC20 ΔKR: Defective in forming the core MCC, abrogating SAC function

• CDC20 ΔDR: Forms the core MCC but cannot inhibit a second CDC20 molecule

• CDC20ΔABBA: Lacks the ABBA-binding motif, affecting kinetochore localization

Experimental Design Considerations:

• For functional studies, RNAi-resistant plasmids expressing wildtype or mutant CDC20 variants should be transfected during cell synchronization

• Fluorescent tagging (YFP, Venus, mCherry) facilitates tracking of exogenous CDC20 expression and localization

• Mitotic progression should be monitored by live-cell imaging following CDC20 manipulation to assess functional consequences

How should CDC20 antibodies be used in ubiquitylation assays to study APC/C activity?

Ubiquitylation assays using CDC20 antibodies provide powerful insights into APC/C activity and regulation:

Reconstituted Ubiquitylation Assay Protocol:

• APC/C Isolation:

  • Deplete endogenous CDC20 using siRNA treatment for 48 hours

  • Isolate APC/C from mitotic HeLa cell extracts using anti-APC3 (AF3.1) antibody immunoprecipitation

• Reaction Components:

  • Resuspend immunoprecipitates in ubiquitylation reaction buffer containing:

    • E1 ligase

    • UbcH10 (E2)

    • Ubiquitin

    • ATP and ATP regenerating system

    • Fluorescently labeled substrate (typically securin)

  • Buffer composition: QA buffer (100 mM NaCl, 30 mM HEPES pH 7.8, 2 mM ATP, 2 mM MgCl₂, 0.1 μg/μl BSA, 1 mM DTT)

  • Supply with recombinant CDC20 and/or core rMCC as indicated

• Substrate Preparation:

  • Label recombinant securin protein with IRDye680 (IRDye 680LT Maleimide Infrared Dye from LI-COR) according to manufacturer's instructions

  • For analyzing ubiquitylation of CDC20, MAD2, and BUBR1, use quantitative immunoblotting with appropriate antibodies

• Detection Method:

  • After SDS-PAGE, directly scan fluorescently labeled substrates using a Li-COR Odyssey CCD scanner

  • For immunoblotting, incubate with fluorescently labeled secondary antibodies and measure fluorescence using the same scanner

Experimental Variables to Consider:

• Time course experiments (10 minutes to several hours) reveal kinetics of APC/C activity and inhibition

• Comparing wildtype CDC20 with mutant variants (ΔDR, ΔKR) elucidates mechanisms of APC/C regulation

• Including core MCC components allows investigation of checkpoint-mediated APC/C inhibition

This assay system provides quantitative measurements of APC/C activity and allows detailed mechanistic studies of how CDC20 and its regulators control cell cycle progression.

How can CDC20 expression levels be leveraged as a biomarker for cancer treatment strategies?

CDC20 expression levels show significant potential as a biomarker for guiding cancer treatment strategies, particularly for therapies targeting the Spindle Assembly Checkpoint:

• Predictor of SAC inhibition sensitivity: High CDC20 expression strongly correlates with increased sensitivity to MPS1 inhibitors and genetic disruption of SAC components . Statistical analyses incorporating CDC20 expression as a covariate completely eliminate the association between aneuploidy and response to SAC inhibitors, suggesting CDC20 is a primary determinant of this sensitivity .

• Association with aneuploidy: Highly aneuploid cancer cells consistently overexpress CDC20 compared to near-diploid cells . This pattern has been observed across diverse cancer cell lines and can be detected at both mRNA and protein levels. The association with aneuploidy provides a mechanistic basis for the potential clinical utility of CDC20 as a biomarker.

• Experimental approaches for biomarker validation:

  • Transcriptomic profiling of tumor samples to quantify CDC20 mRNA levels

  • Immunohistochemistry using validated CDC20 antibodies to assess protein expression in patient samples

  • Correlation of expression levels with response to SAC-targeting therapeutics in patient-derived xenograft models

• Potential clinical applications:

  • Stratification of patients for clinical trials of MPS1 inhibitors and other SAC-targeting drugs

  • Development of companion diagnostics for SAC inhibition therapy

  • Personalized treatment planning based on CDC20 expression profiles in individual tumors

This evidence suggests that CDC20 expression could serve as a molecular feature associated with sensitivity to SAC inhibition therapy and as a potential biomarker for patient selection .

What challenges exist in distinguishing CDC20 functional isoforms using antibody-based approaches?

Several significant challenges complicate the use of antibody-based approaches for distinguishing CDC20 functional isoforms:

• Multiple protein isoforms: Recent research has identified several CDC20 protein isoforms that may affect mitotic duration . These isoforms likely result from alternative splicing, post-translational modifications, or alternative translation start sites, creating variants with potentially distinct functional properties.

• Epitope accessibility issues: Depending on protein conformation and interaction status, antibody epitopes may be masked or exposed differently across isoforms. This can lead to inconsistent detection of specific isoforms depending on their cellular context or binding partners.

• Post-translational modification interference: CDC20 undergoes various post-translational modifications including phosphorylation and ubiquitylation . These modifications may alter antibody binding efficiency or specificity, potentially causing differential detection of modified isoforms.

• Cross-reactivity concerns: Antibodies developed against one CDC20 isoform may cross-react with other isoforms due to shared sequence homology, complicating specific isoform quantification.

• Methodological approaches to address these challenges:

  • Use of multiple antibodies targeting different CDC20 epitopes

  • Two-dimensional gel electrophoresis to separate isoforms based on both molecular weight and isoelectric point before immunodetection

  • Mass spectrometry-based approaches for isoform identification and quantification

  • Expression of tagged isoform-specific constructs for validation of antibody specificity

Despite these challenges, current research suggests that the relative abundance of CDC20 isoforms does not differ significantly between diploid and aneuploid cells , suggesting that total CDC20 levels may be more relevant than isoform distribution in certain contexts.

How do CDC20 interactions with the Mitotic Checkpoint Complex change under different cellular stresses?

The dynamic interactions between CDC20 and the Mitotic Checkpoint Complex (MCC) undergo significant changes in response to various cellular stresses:

• Microtubule-targeting agents:

  • Nocodazole (complete microtubule depolymerization) induces strong SAC activation, leading to robust MCC-CDC20 complex formation and sustained mitotic arrest

  • Taxol (microtubule stabilization) activates a weaker SAC response compared to nocodazole, resulting in less stable MCC-CDC20 interactions

  • These different responses highlight the sensitivity of CDC20-MCC interactions to the nature and severity of spindle disruption

• Experimental approaches to study these dynamic interactions:

  • Size exclusion chromatography can separate different CDC20-containing complexes based on molecular size

  • Immunoprecipitation with antibodies against different MCC components (MAD2, BUBR1) followed by CDC20 detection can reveal stress-specific complex formation patterns

  • Live-cell imaging with fluorescently tagged proteins allows real-time monitoring of complex formation and disassembly

  • FRET-based approaches can detect direct interactions between CDC20 and MCC components under different stress conditions

• Mechanistic considerations:

  • The core MCC (MAD2, BUBR1, CDC20 in 1:1:1 ratio) inhibits a second CDC20 molecule that has already bound and activated the APC/C

  • Under prolonged mitotic arrest, CDC20 itself becomes ubiquitylated, potentially altering MCC binding dynamics

  • Different stresses may affect the relative importance of various CDC20 interaction domains (KEN-box recognition, D-box recognition) in MCC binding and APC/C inhibition

Understanding these dynamic interactions is crucial for developing more effective strategies to manipulate the SAC in cancer therapy and for explaining differential cellular responses to various anti-mitotic agents.

What experimental approaches can effectively distinguish between CDC20's mitotic and transcriptional regulatory functions?

Distinguishing between CDC20's mitotic progression and transcriptional regulatory functions requires sophisticated experimental approaches that can separate these temporally and mechanistically distinct activities:

• Domain-specific mutant analysis:

  • The WD40 domain of CDC20 is required for its transcriptional regulatory functions

  • Generate and express domain-specific mutants that selectively disrupt either mitotic or transcriptional functions

  • For example, mutants that maintain APC/C interaction but disrupt binding to transcriptional machinery components

• Temporal separation approaches:

  • Synchronize cells in G1/S using double thymidine block, then release and collect samples at various time points

  • Analyze transcriptional targets (e.g., UbcH10) before cells enter mitosis

  • Use CDC20 inhibition specifically during interphase to assess transcriptional effects independent of mitotic functions

• Subcellular localization studies:

  • Perform fractionation experiments to separate nuclear and cytoplasmic CDC20 pools

  • Utilize immunofluorescence microscopy with co-staining for transcriptional machinery components versus mitotic apparatus

  • Develop CDC20 variants with altered nuclear localization signals to preferentially direct CDC20 to transcriptional sites

• Protein interaction network analysis:

  • Perform immunoprecipitation coupled with mass spectrometry at different cell cycle stages

  • Compare CDC20 interactomes during interphase (when transcriptional regulation predominates) versus mitosis

  • Identify distinct interaction partners mediating transcriptional versus mitotic functions

• Chromatin association studies:

  • Conduct ChIP-seq experiments to identify CDC20 binding sites on chromatin

  • Perform sequential ChIP to detect CDC20 co-occupancy with known transcriptional regulators like CBP

  • Correlate binding patterns with expression changes of target genes like UbcH10

These approaches provide complementary information that, when integrated, can effectively distinguish between CDC20's dual roles in mitotic progression and transcriptional regulation.

How can researchers address the challenge of detecting residual CDC20 in knockout models?

The challenge of detecting residual CDC20 in presumed knockout models requires sophisticated approaches that go beyond standard western blotting:

• Mass spectrometry-based detection:

  • Immunoprecipitate using antibodies against CDC20 C-terminus

  • Perform liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis on the immunoprecipitate

  • This approach has successfully detected CDC20 peptides in presumed knockout clones where western blot signals were below detection threshold

• Functional readouts of residual CDC20:

  • Monitor mitotic duration in presumed knockout clones (extended mitosis indicates partial CDC20 depletion)

  • Test sensitivity to CDC20 RNAi (true knockouts would be insensitive)

  • Perform rescue experiments with RNAi-resistant CDC20 constructs

• Amplification techniques for protein detection:

  • Use proximity ligation assays (PLA) to detect CDC20 interactions with known binding partners

  • Employ tyramide signal amplification in immunofluorescence approaches

  • Utilize highly sensitive chemiluminescent substrates for western blotting

• Protein enrichment strategies:

  • Synchronize cells at prometaphase to maximize CDC20 expression

  • Use larger amounts of starting material for immunoprecipitation

  • Employ tandem immunoprecipitation to increase specificity and concentration

Complete CDC20 knockout typically causes metaphase arrest and apoptosis , so the survival of presumed knockout clones strongly suggests the presence of residual protein. These approaches can help researchers accurately characterize their model systems and avoid misinterpretation of experimental results.

What controls are essential when using CDC20 antibodies for quantitative applications?

Robust quantitative applications of CDC20 antibodies require comprehensive controls to ensure reliability and reproducibility:

• Expression level controls:

  • Cell cycle synchronization: Essential due to CDC20's fluctuating expression throughout the cell cycle. Compare samples only at matching cell cycle stages, ideally using nocodazole synchronization for prometaphase arrest

  • Loading controls: Use multiple loading controls (tubulin, GAPDH) and normalization to total protein (Ponceau S staining)

  • Dynamic range validation: Create a standard curve using defined amounts of recombinant CDC20 to ensure measurements fall within the linear range of detection

• Specificity controls:

  • Partial knockdown samples: Include CDC20 siRNA/shRNA-treated samples as specificity controls

  • Competing peptide: Pre-incubate antibody with excess immunizing peptide to demonstrate binding specificity

  • Multiple antibodies: Validate key findings using antibodies targeting different CDC20 epitopes

• Technical controls for specific applications:

  • Western blotting: Include both positive controls (cells overexpressing CDC20) and negative controls (CDC20-depleted cells)

  • Immunofluorescence: Perform peptide competition and secondary-only controls; validate localization patterns across multiple cell lines

  • Immunoprecipitation: Use IgG controls and validate with reciprocal IP approaches

  • ChIP assays: Include IgG controls and validate with multiple primer sets

• Quantification methodology:

  • For western blots, use fluorescently labeled secondary antibodies and direct scanning (LI-COR Odyssey) rather than chemiluminescence for more accurate quantification

  • For immunofluorescence, standardize image acquisition parameters and use automated analysis algorithms to avoid bias

Implementation of these comprehensive controls ensures that quantitative measurements of CDC20 accurately reflect biological reality rather than technical artifacts.

How can non-specific binding issues be mitigated when using CDC20 antibodies in complex samples?

Non-specific binding presents a significant challenge when using CDC20 antibodies, particularly in complex samples such as tissue lysates or heterogeneous cell populations. Several strategies can effectively mitigate these issues:

• Optimization of blocking conditions:

  • Test multiple blocking agents (BSA, milk, commercial blockers) to identify optimal formulation

  • Extended blocking times (2-4 hours) may reduce background in problematic samples

  • Consider adding 0.1-0.5% Triton X-100 to blocking buffer to reduce hydrophobic interactions

• Sample preparation refinements:

  • Pre-clear lysates with Protein A/G beads before immunoprecipitation

  • Include competitive binding agents (0.1-0.5% BSA) in wash buffers

  • For tissue samples, perform additional centrifugation steps to remove insoluble material

• Antibody optimization strategies:

  • Titrate antibody concentration to determine minimal effective concentration

  • Use affinity-purified antibodies rather than crude serum

  • Consider Fab or F(ab')₂ fragments for reduced non-specific binding

  • Crosslink antibodies to beads for immunoprecipitation to prevent heavy/light chain interference on western blots

• Detection system modifications:

  • For western blotting, use highly specific fluorescent secondary antibodies rather than HRP-conjugated antibodies

  • In immunofluorescence, include an extra washing step with high-salt buffer (300-500 mM NaCl)

  • Consider spectral imaging and linear unmixing to distinguish specific signal from autofluorescence

• Validation approaches:

  • Perform parallel experiments with multiple CDC20 antibodies targeting different epitopes

  • Include competitive peptide controls to confirm specificity

  • Use CDC20-depleted samples as negative controls to identify non-specific bands or signals

These strategies, when implemented appropriately for each experimental context, can significantly improve signal-to-noise ratio and enhance the reliability of CDC20 antibody applications in complex biological samples.