Phospho-CDC25C (Ser216) Antibody

What is CDC25C and what role does phosphorylation at Ser216 play in cell cycle regulation?

CDC25C is a dual-specificity phosphatase belonging to the CDC25 phosphatase family that plays a crucial role in cell division regulation. It primarily functions by dephosphorylating cyclin B-bound CDC2 (CDK1), triggering entry into mitosis. Phosphorylation at Ser216 creates a binding site for 14-3-3 proteins, which induces CDC25C export from the nucleus during interphase in response to DNA damage . This nuclear export mechanism prevents premature mitotic entry when DNA integrity is compromised, serving as a critical cell cycle checkpoint. The phosphorylation state of CDC25C at Ser216, therefore, regulates its subcellular localization and ultimately its ability to activate cyclin B-CDK1 complexes.

How does phosphorylated CDC25C (Ser216) interact with other cell cycle checkpoint proteins?

Phosphorylated CDC25C at Ser216 serves as a docking site for 14-3-3 proteins, resulting in cytoplasmic sequestration of CDC25C. This interaction forms part of a larger network of checkpoint proteins that respond to DNA damage signals. When DNA damage occurs, checkpoint kinases like CHK1, CHK2, and other kinases phosphorylate CDC25C at Ser216, promoting 14-3-3 binding and nuclear export . This keeps CDC25C away from its nuclear substrates, preventing cell cycle progression until DNA repair is completed. Additionally, phospho-CDC25C (Ser216) has been shown to integrate signals from multiple pathways including ATR/CHK1, ATM/CHK2, CHK2/ERK, and JNK/p38 signaling cascades , highlighting its central role in coordinating cellular responses to stress.

What are the optimal dilution ratios for phospho-CDC25C (Ser216) antibody across different applications?

Based on published protocols and manufacturer recommendations, the optimal working dilutions for phospho-CDC25C (Ser216) antibody vary by application:

It's important to note that these are starting recommendations, and optimal dilutions may need to be determined empirically for each specific experimental setup. When establishing a new protocol, it's advisable to test a range of dilutions to determine the optimal signal-to-noise ratio for your specific sample type and detection system .

How can researchers validate the specificity of phospho-CDC25C (Ser216) antibody to ensure it doesn't cross-react with other CDC25 isoforms?

Validating specificity of phospho-CDC25C (Ser216) antibody is critical for accurate interpretation of results. Several approaches can be implemented:

• Peptide competition assay: Incubate the antibody with phospho-CDC25C (Ser216) peptide before application to samples. This should abolish specific signal, as demonstrated in published validation studies .

• Phosphatase treatment: Treat half of your sample with calf intestinal alkaline phosphatase (CIAP). The phospho-CDC25C (Ser216) antibody should not detect the dephosphorylated protein, confirming phospho-specificity .

• Kinase and phosphatase assays: These can be performed to validate that the antibody exclusively detects the phosphorylated form. Research has shown that anti-phospho-CDC25C (Ser216) fails to detect proteins treated with CIAP, confirming its specificity for the phosphorylated form .

• Cross-reactivity testing: Studies have demonstrated that phospho-CDC25C (Ser216) antibody does not cross-react with phosphorylated CDC25B, as confirmed by both biochemical assays and immunohistochemical analysis of serial tissue sections .

• siRNA knockdown: Comparing antibody staining in control versus CDC25C-knockdown cells provides definitive evidence of specificity.

These validation steps ensure that observed signals are truly representative of phosphorylated CDC25C at Ser216, not other phosphorylated residues or related phosphatases.

What are effective sample preparation methods for optimal detection of phospho-CDC25C (Ser216) in cellular lysates?

For optimal detection of phospho-CDC25C (Ser216) in cellular lysates, several critical sample preparation steps should be followed:

• Rapid sample processing: Phosphorylation states can change rapidly after cell lysis. Process samples immediately on ice to preserve phosphorylation status.

• Phosphatase inhibitors: Include a comprehensive phosphatase inhibitor cocktail in lysis buffers (e.g., sodium fluoride, sodium orthovanadate, β-glycerophosphate, and sodium pyrophosphate) to prevent dephosphorylation during processing.

• Protease inhibitors: Add protease inhibitors to prevent degradation of CDC25C protein.

• Lysis buffer composition: Use a buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, supplemented with the inhibitors mentioned above.

• Sample volume optimization: For techniques like AlphaLISA SureFire Ultra detection, a sample volume of 10 μL is recommended based on established protocols .

• Positive controls: Include samples from cells treated with DNA damaging agents (e.g., hydroxyurea, etoposide) or checkpoint activators, which increase Ser216 phosphorylation.

These preparation steps have been validated in research contexts to maintain phosphorylation status and ensure reliable detection of phospho-CDC25C (Ser216) .

How can phospho-CDC25C (Ser216) antibody be effectively used in immunohistochemistry for tissue samples?

For effective immunohistochemical detection of phospho-CDC25C (Ser216) in tissue samples, researchers should follow these validated protocols:

• Fixation and embedding: Use formalin-fixed, paraffin-embedded tissue sections. Overfixation can mask epitopes, so standardize fixation times.

• Antigen retrieval: Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) for 20 minutes to expose the phospho-epitope.

• Blocking endogenous peroxidase: Incubate sections in 3% H₂O₂ for 5 minutes to reduce background.

• Primary antibody incubation: Apply phospho-CDC25C (Ser216) antibody at a 1:50-1:100 dilution and incubate overnight at 4°C for optimal results .

• Detection system: Use a sensitive detection system such as Advance™ HRP link followed by Advance HRP enzyme incubation for 30 minutes each .

• Visualization: Develop with 3'3-diaminobenzidine tetrahydrochloride (DAB) for 10 minutes, followed by hematoxylin counterstaining .

• Controls: Include positive controls (breast carcinoma or tonsil tissue with known phospho-CDC25C expression) . For negative controls, substitute the primary antibody with mouse myceloma protein of the same subclass and concentration, or use the antibody pre-absorbed with phospho-CDC25C (Ser216) peptide.

This protocol has been successfully employed in research studies examining phospho-CDC25C (Ser216) expression in carcinomas and other tissues .

What scoring systems are used to quantify phospho-CDC25C (Ser216) expression in immunohistochemical studies?

In published research on phospho-CDC25C (Ser216), several validated scoring systems have been employed for immunohistochemical quantification:

• Combined score system: A scoring system that evaluates both staining intensity and percentage of positive cells has been effectively used in studies of vulvar carcinomas . This system calculates a total score by multiplying intensity (0-3) by percentage (0-3), resulting in scores ranging from 0-9.

• Subcellular localization distinction: Given that phospho-CDC25C (Ser216) can localize to different cellular compartments, separate scoring for cytoplasmic (score >3 considered high) and nuclear (score >0 considered high) staining provides valuable functional information .

• Combined cytoplasmic/nuclear assessment: Some studies have benefited from a combined assessment where high phospho-CDC25C (Ser216) immunostaining is defined as a score >3 when accounting for both compartments .

In research applications, selection of the appropriate scoring system should be based on the specific research question, particularly if investigating the relationship between subcellular localization of phospho-CDC25C and biological outcomes.

What are common technical issues in phospho-CDC25C (Ser216) detection and how can they be resolved?

Researchers commonly encounter several technical challenges when working with phospho-CDC25C (Ser216) antibodies:

• High background signal:

  • Cause: Insufficient blocking or non-specific binding

  • Solution: Increase blocking time (5% BSA or normal serum), optimize antibody dilution, and include 0.1% Tween-20 in wash buffers

• Weak or absent signal:

  • Cause: Rapid dephosphorylation during sample preparation

  • Solution: Ensure immediate sample processing on ice and include adequate phosphatase inhibitors in all buffers

• False negative results:

  • Cause: Epitope masking during fixation (particularly relevant for IHC)

  • Solution: Optimize antigen retrieval methods; test both citrate and EDTA-based retrieval solutions

• Inconsistent results between experiments:

  • Cause: Variations in cell cycle distribution or stress conditions

  • Solution: Standardize cell culture conditions and synchronize cells when appropriate

• Multiple bands in Western blot:

  • Cause: Detection of alternatively spliced variants or degradation products

  • Solution: Include positive controls with known phospho-CDC25C (Ser216) expression; validate bands using siRNA knockdown or phosphatase treatment

These troubleshooting approaches have been validated in published methodological studies and can significantly improve detection specificity and reproducibility.

How does phospho-CDC25C (Ser216) expression correlate with cancer progression and patient outcomes?

Research has revealed significant correlations between phospho-CDC25C (Ser216) expression and cancer characteristics:

• Association with aggressive tumor phenotypes: In a comprehensive study of 300 vulvar carcinomas, high phospho-CDC25C (Ser216) expression significantly correlated with:

  • High FIGO stage

  • Large tumor diameter

  • Deep invasion

  • Poor differentiation

• Subcellular distribution patterns: High phospho-CDC25C (Ser216) expression was identified in:

  • Cytoplasm (50% of cases)

  • Nucleus (70% of cases)

  • Combined cytoplasm/nucleus (77% of cases)

• Survival analysis correlations: Univariate analysis revealed that high expression of phospho-CDC25C (Ser216) correlated with poor disease-specific survival (p = 0.04), though this association was not maintained in multivariate analysis .

• Potential as a biomarker: While phospho-CDC25C (Ser216) shows significant associations with malignant features, research suggests it may be more valuable as part of a panel of markers rather than as an independent prognostic indicator. Further studies are needed to clarify its role as a prognostic marker .

These findings suggest phospho-CDC25C (Ser216) plays a crucial role in the pathogenesis and progression of certain carcinomas, though its independent prognostic value requires further investigation.

What is the relationship between DNA damage response pathways and phospho-CDC25C (Ser216) levels?

The relationship between DNA damage response pathways and phospho-CDC25C (Ser216) levels is complex and bidirectional:

• Checkpoint kinase activation: DNA damage activates checkpoint kinases (CHK1, CHK2) which directly phosphorylate CDC25C at Ser216, creating a binding site for 14-3-3 proteins .

• Subcellular sequestration mechanism: Phosphorylated CDC25C (Ser216) bound to 14-3-3 proteins is exported from the nucleus to the cytoplasm, preventing premature mitotic entry in the presence of DNA damage .

• Integration of multiple stress signals: Research has shown that phospho-CDC25C (Ser216) serves as an integration point for signals from various pathways including ATR/CHK1, ATM/CHK2, and stress-activated kinase pathways (JNK/p38) .

• Cell cycle-dependent regulation: Studies have demonstrated that CDC25C phosphatase activity changes during cell cycle progression, with interphase cells showing basal activity that increases during the G2/M transition. This is accompanied by changes in phospho-CDC25C (Ser216) levels and its interaction with other proteins .

• Role in G2/M checkpoint-mediated apoptosis: Evidence suggests phospho-CDC25C (Ser216) may play a role in determining cell fate (repair vs. apoptosis) following DNA damage through its interactions with pro-apoptotic proteins like ASK1 (apoptosis signal-regulating kinase 1) .

These insights highlight the central role of phospho-CDC25C (Ser216) in coordinating cellular responses to genotoxic stress and maintaining genomic integrity.

How do post-translational modifications beyond Ser216 phosphorylation affect CDC25C function and antibody detection?

CDC25C undergoes multiple post-translational modifications that interact with Ser216 phosphorylation and can impact antibody detection:

• Multiple phosphorylation sites: CDC25C contains numerous phosphorylation sites beyond Ser216, including sites targeted by CDK1, PLK1, and Aurora kinases during mitotic entry. These can affect protein conformation and potentially mask the Ser216 epitope or alter antibody accessibility .

• Hyperphosphorylation states: During mitotic arrest, CDC25C becomes hyperphosphorylated at multiple sites, exhibiting enhanced phosphatase activity but significantly reduced affinity to certain binding partners such as ASK1 . This state can potentially alter epitope presentation for antibody detection.

• Phosphorylation-dependent protein interactions: Research has shown that Ser216 phosphorylation creates a binding site for 14-3-3 proteins, which can shield the phospho-epitope from antibody recognition in certain contexts .

• Other modifications: Beyond phosphorylation, CDC25C undergoes other modifications including ubiquitination and SUMOylation, which can affect protein stability, localization, and epitope accessibility.

When designing experiments, researchers should consider how different cell cycle phases and cellular stresses might alter the post-translational modification landscape of CDC25C, potentially affecting detection by phospho-specific antibodies. Validation experiments under various cellular conditions are recommended for comprehensive interpretation.

What are the differences in CDC25C isoform expression and Ser216 phosphorylation across different tissue types and disease states?

Research has revealed significant heterogeneity in CDC25C isoform expression and Ser216 phosphorylation patterns:

• Tissue-specific expression patterns: While CDC25C is widely expressed, its expression levels and isoform distribution vary considerably across tissues. Studies have shown particular relevance in rapidly dividing tissues and those undergoing regulated cell cycle control .

• Cancer-specific alterations: In vulvar carcinomas, high cytoplasmic CDC25C expression was observed in 63% of cases, while phospho-CDC25C (Ser216) showed a more complex distribution pattern with high expression in cytoplasm (50%), nucleus (70%), and combined compartments (77%) .

• Isoform heterogeneity: Multiple alternatively spliced transcript variants of CDC25C have been described, but their full-length nature and functional significance remain incompletely characterized . This diversity may contribute to differential phosphorylation patterns and antibody reactivity.

• Subcellular localization differences: The subcellular distribution of phospho-CDC25C (Ser216) varies significantly across cell types and disease states. While predominantly cytoplasmic in normal cells (due to 14-3-3 binding), altered distribution patterns are observed in cancer cells, potentially reflecting dysregulated checkpoint mechanisms .

• Correlation with malignant features: Research has demonstrated significant associations between phospho-CDC25C (Ser216) expression and malignant features in certain cancers, including correlations with tumor stage, differentiation status, invasion depth, and nodal metastasis .

These variations highlight the importance of tissue-specific and context-dependent analysis when studying phospho-CDC25C (Ser216) in different biological systems or disease models.

What experimental approaches can be used to study the dynamic regulation of CDC25C phosphorylation at Ser216 during the cell cycle?

Advanced experimental approaches to study dynamic regulation of CDC25C phosphorylation at Ser216 include:

• Live-cell imaging with phospho-specific biosensors:

  • Development of FRET-based biosensors incorporating the CDC25C phospho-binding domain of 14-3-3 proteins

  • Enables real-time visualization of phosphorylation/dephosphorylation events in living cells

• Synchronization protocols for cell cycle analysis:

  • Double thymidine block for G1/S boundary synchronization

  • Nocodazole treatment for mitotic arrest

  • Sequential sampling to track phospho-CDC25C (Ser216) levels throughout cell cycle progression

• Quantitative phosphoproteomics:

  • SILAC or TMT-based approaches to quantify changes in CDC25C phosphorylation across multiple sites simultaneously

  • Correlation of Ser216 phosphorylation with other post-translational modifications

• In vitro kinase and phosphatase assays:

  • Recombinant protein-based assays to identify kinases and phosphatases that regulate Ser216 phosphorylation

  • Phospho-specific antibodies can be used to monitor these reactions

• Cellular stress response studies:

  • Treatment with DNA damaging agents (e.g., hydroxyurea, etoposide) to activate checkpoint kinases

  • Time-course analysis of phospho-CDC25C (Ser216) levels using techniques like AlphaLISA SureFire Ultra assays

• Protein-protein interaction studies:

  • Co-immunoprecipitation experiments to identify phosphorylation-dependent interactors

  • Proximity ligation assays to visualize interactions with 14-3-3 proteins in situ

These approaches provide complementary information about the spatiotemporal regulation of CDC25C phosphorylation at Ser216 under various physiological and pathological conditions.

How might targeting phospho-CDC25C (Ser216) pathways contribute to cancer therapeutic strategies?

Emerging research suggests several promising approaches for targeting phospho-CDC25C (Ser216) pathways in cancer therapy:

• Checkpoint kinase modulators: Compounds that enhance CHK1/CHK2 activity could increase CDC25C phosphorylation at Ser216, promoting its cytoplasmic sequestration and preventing premature mitotic entry in cancer cells with compromised G2/M checkpoints .

• 14-3-3 interaction stabilizers: Small molecules that stabilize the interaction between phospho-CDC25C (Ser216) and 14-3-3 proteins could enhance cytoplasmic retention, potentially sensitizing cancer cells to DNA-damaging therapies.

• Combination with conventional chemotherapeutics: Studies suggest that modulating phospho-CDC25C (Ser216) levels might enhance the efficacy of conventional DNA-damaging agents by preventing checkpoint recovery and promoting apoptosis in cancer cells .

• Synthetic lethality approaches: Since phospho-CDC25C (Ser216) plays a crucial role in the DNA damage response, targeting this pathway might be particularly effective in cancers with defects in complementary DNA repair mechanisms.

• Biomarker-guided therapy selection: Assessment of phospho-CDC25C (Ser216) levels and localization patterns might help identify tumors likely to respond to checkpoint-targeted therapies or conventional DNA-damaging agents .

While these approaches show promise, challenges remain in developing specific modulators of this pathway and understanding the consequences of such interventions in different cellular contexts and cancer types.

What methodological innovations might improve quantitative analysis of phospho-CDC25C (Ser216) in complex biological samples?

Several innovative methodological approaches are emerging to enhance phospho-CDC25C (Ser216) detection and quantification:

• Single-cell phosphoproteomics:

  • Emerging technologies for single-cell analysis of phosphorylation states

  • Provides insights into cell-to-cell variability in phospho-CDC25C (Ser216) levels within heterogeneous populations

• Multiplexed immunoassays:

  • Development of multiplexed detection systems like AlphaLISA SureFire Ultra platforms that allow simultaneous quantification of phospho-CDC25C (Ser216) and related signaling molecules

  • Enables comprehensive pathway analysis from limited sample volumes

• Digital pathology and AI-assisted image analysis:

  • Machine learning algorithms for automated quantification of phospho-CDC25C (Ser216) immunohistochemical staining

  • Improves reproducibility and allows detection of subtle patterns not apparent by manual scoring

• Proximity-based detection methods:

  • Proximity ligation assays (PLA) to visualize and quantify interactions between phospho-CDC25C (Ser216) and binding partners like 14-3-3 proteins

  • Provides spatial information about these interactions within cells

• Mass spectrometry-based absolute quantification:

  • Development of AQUA (Absolute Quantification) peptides for phospho-CDC25C (Ser216)

  • Enables precise determination of phosphorylation stoichiometry