Phospho-ATRIP (S224) Antibody

What is ATRIP and why is the S224 phosphorylation site significant?

ATRIP (ATR interacting protein) is an essential component of the DNA damage checkpoint pathway. The protein binds to single-stranded DNA coated with replication protein A and interacts with the ataxia telangiectasia and Rad3 related protein kinase (ATR), resulting in accumulation at intranuclear foci induced by DNA damage . The S224 phosphorylation site is particularly significant because it represents a regulatory node where cell cycle control intersects with DNA damage response. S224 is phosphorylated in a cell cycle-dependent manner by CDK2-cyclin A, and this phosphorylation regulates the ability of ATR-ATRIP to promote cell cycle arrest in response to DNA damage . Mutation of S224 to alanine causes a defect in the ATR-ATRIP-dependent maintenance of the G2-M checkpoint to ionizing and UV radiation .

Which experimental techniques can effectively detect ATRIP S224 phosphorylation?

Multiple experimental approaches can be employed to detect ATRIP S224 phosphorylation:

• Western Blotting (WB): Using phospho-specific antibodies at dilutions of 1:500-1:2000 . This technique allows quantitative analysis of phosphorylation levels in cell lysates.

• Immunohistochemistry (IHC): Recommended dilutions are 1:100-1:300 . This approach enables visualization of S224 phosphorylation in tissue sections or fixed cells.

• ELISA: Can be performed at dilutions up to 1:10000 . The ATRIP Phospho-Ser224 Colorimetric Cell-Based ELISA Kit offers a high-throughput approach for measuring relative amounts of phosphorylated ATRIP in cultured cells .

• Mass Spectrometry: This technique was originally used to identify the S224 phosphorylation site and can be employed for unbiased phosphoproteomic analysis .

How should phospho-ATRIP (S224) antibodies be stored and handled?

Proper storage and handling of phospho-ATRIP (S224) antibodies is crucial for maintaining their specificity and sensitivity:

• Storage Temperature: Store at -20°C for long-term preservation . Some antibodies may be shipped at 4°C but should be aliquoted and stored at -20°C or -80°C upon delivery .

• Buffer Composition: Typically maintained in PBS containing 50% glycerol, 0.5% BSA, and 0.02% sodium azide .

• Freeze-Thaw Cycles: Avoid repeated freeze-thaw cycles as this can degrade antibody quality .

• Working Solutions: Prepare fresh dilutions in appropriate buffers according to the application (WB, IHC, or ELISA).

• Validation: Prior to experimental use, validate antibody specificity using positive controls (cells with known S224 phosphorylation) and negative controls (phosphatase-treated samples or S224A mutant cells).

How does CDK2-dependent phosphorylation of ATRIP at S224 mechanistically regulate the DNA damage checkpoint?

CDK2-dependent phosphorylation of ATRIP at S224 plays a specific role in regulating the ATR-ATRIP DNA damage response pathway:

• Cell Cycle Coordination: S224 phosphorylation occurs in a cell cycle-dependent manner, suggesting it coordinates DNA damage responses with cell cycle progression . This provides a mechanism for differential checkpoint activation depending on cell cycle phase.

• Checkpoint Maintenance: While S224 phosphorylation is not required for ATRIP binding to RPA, localization to sites of DNA damage, or binding to ATR, it is specifically required for maintaining the G2-M checkpoint following ionizing and UV radiation . This suggests it regulates downstream signaling events rather than initial damage recognition.

• Integration with CDK Signaling: Since S224 is phosphorylated by CDK2-cyclin A, this site represents a regulatory node where cell cycle kinase activity directly impacts DNA damage checkpoint function . This creates a feedback loop where cell cycle position influences damage response capacity.

• Substrate Recognition: S224 phosphorylation may alter ATRIP conformation or create binding sites for other factors that enhance ATR kinase activity toward specific substrates involved in checkpoint maintenance.

What experimental considerations are important when studying the relationship between cell cycle progression and ATRIP S224 phosphorylation?

When investigating the relationship between cell cycle progression and ATRIP S224 phosphorylation, researchers should consider:

• Cell Synchronization Methods: Different synchronization techniques (serum starvation, thymidine block, nocodazole arrest) may affect the baseline phosphorylation status of ATRIP. Researchers should validate S224 phosphorylation patterns across multiple synchronization methods.

• CDK2 Activity Monitoring: Since S224 is phosphorylated by CDK2-cyclin A, parallel assessment of CDK2 activity (using histone H1 kinase assays or other CDK2 substrates) provides context for interpreting S224 phosphorylation data .

• Cell Cycle Markers: Co-staining for cell cycle markers (cyclin A, cyclin B, phospho-histone H3) in immunofluorescence experiments or parallel flow cytometry can precisely correlate S224 phosphorylation with cell cycle phase.

• CDK Inhibitors: Careful titration of CDK inhibitors is necessary, as complete inhibition may trigger checkpoint responses that confound interpretation of S224 phosphorylation effects .

• Phosphomimetic and Phosphodeficient Mutants: Complementation experiments with S224A (phosphodeficient) and S224D/E (phosphomimetic) mutants can help distinguish between effects of phosphorylation timing versus the simple presence/absence of the phosphate group.

How do other ATRIP post-translational modifications interact with S224 phosphorylation?

ATRIP undergoes multiple post-translational modifications that may interact with S224 phosphorylation:

• Additional Phosphorylation Sites: In addition to S224, S239 was identified as another phosphorylation site . Researchers should investigate potential priming relationships, where phosphorylation at one site influences modification at another.

• Hierarchical Phosphorylation: While S224 is modified by CDK2, other ATRIP residues may be targeted by additional kinases including ATR itself, creating potential feedback loops in the signaling pathway .

• Potential Cross-talk: Experimental designs should consider how:

  • Phosphorylation may influence other modifications (ubiquitination, SUMOylation)

  • DNA damage-induced modifications may enhance or antagonize cell cycle-dependent modifications

  • Temporal sequences of modifications may determine signaling outcomes

• Oligomerization Effects: Since ATR-ATRIP may form higher-order oligomeric complexes, S224 phosphorylation could influence complex assembly or stability . Co-immunoprecipitation experiments under various conditions can address this possibility.

What are the optimal conditions for detecting ATRIP S224 phosphorylation by Western blot?

For optimal detection of ATRIP S224 phosphorylation by Western blot, consider the following protocol elements:

• Cell Lysis and Phosphatase Inhibition:

  • Use buffers containing 50mM Tris-HCl pH 7.5, 150mM NaCl, 1% NP-40 or Triton X-100

  • Include phosphatase inhibitors (10mM NaF, 1mM Na3VO4, 10mM β-glycerophosphate)

  • Add protease inhibitor cocktail and maintain samples at 4°C throughout processing

• Sample Preparation:

  • Use fresh samples when possible

  • For SDS-PAGE, load 20-50μg total protein per lane

  • Include positive controls (asynchronous cells) and negative controls (λ-phosphatase treated samples)

• Antibody Conditions:

  • Primary antibody dilution: 1:500-1:2000

  • Incubation: Overnight at 4°C with gentle agitation

  • Blocking: 5% BSA in TBST (preferred over milk which contains phosphatases)

  • Secondary antibody: HRP-conjugated anti-rabbit IgG at 1:5000-1:10000

• Detection System:

  • Enhanced chemiluminescence (ECL) systems with extended exposure times

  • Consider using signal enhancer solutions for low abundance phosphoproteins

• Normalization and Controls:

  • Reprobe membranes for total ATRIP to calculate phospho/total ratios

  • Include cell cycle markers (cyclin A, B) to correlate with cell cycle phase

  • Use CDK2 inhibitor-treated samples as specificity controls

What protocols are recommended for immunohistochemical detection of phospho-ATRIP (S224)?

For effective immunohistochemical detection of phospho-ATRIP (S224), implement these protocol considerations:

• Tissue Preparation:

  • Fix tissues in 10% neutral buffered formalin for 24 hours

  • Embed in paraffin and section at 4-5μm thickness

  • Mount on positively charged slides

• Antigen Retrieval:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Pressure cooker treatment for 15-20 minutes is recommended for phospho-epitopes

• Blocking and Antibody Incubation:

  • Block endogenous peroxidase with 3% H2O2 in methanol

  • Block non-specific binding with 5% normal goat serum

  • Use phospho-ATRIP (S224) antibody at 1:100-1:300 dilution

  • Incubate overnight at 4°C in a humidified chamber

• Detection System:

  • Use polymer-based detection systems for enhanced sensitivity

  • Develop with DAB substrate and counterstain with hematoxylin

  • Mount with permanent mounting medium

• Controls and Validation:

  • Include positive control tissues (proliferating tissues with DNA damage)

  • Use phosphatase-treated serial sections as negative controls

  • Consider dual immunofluorescence with cell cycle markers to correlate phosphorylation with cell cycle phase

How can ELISA be optimized for quantitative analysis of ATRIP S224 phosphorylation?

For optimized ELISA-based quantitative analysis of ATRIP S224 phosphorylation, implement these technical approaches:

• Cell-Based ELISA Protocol:

  • Seed cells at consistent density (typically >5000 cells per well)

  • Perform treatments in 96-well plates

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% BSA in PBS

• Antibody Applications:

  • Use phospho-ATRIP (S224) primary antibody at 1:10000 dilution

  • Employ HRP-conjugated secondary antibody

  • Develop with TMB substrate and measure absorbance at 450nm

• Data Normalization Strategies:

  • Normalize phospho-signal to total ATRIP

  • Alternatively, normalize to cell number using DNA-binding dyes

  • Include standard curves using known phosphopeptide concentrations

• Screening Applications:

  • The cell-based ELISA format is particularly suited for screening effects of:

    • siRNA knockdowns

    • Chemical inhibitors

    • DNA damaging agents

    • Cell cycle modulators

• Indirect ELISA Format:

  • For maximum sensitivity, use the indirect format where ATRIP is captured by primary antibody

  • This approach enables detection of low abundance phosphorylated protein

How should researchers interpret ATRIP S224 phosphorylation patterns in relation to DNA damage response?

When interpreting ATRIP S224 phosphorylation patterns in DNA damage response experiments:

• Temporal Dynamics: Analyze the kinetics of S224 phosphorylation following DNA damage. The maintenance phase of the G2-M checkpoint (6-24 hours post-damage) is particularly dependent on S224 phosphorylation .

• Cell Cycle Context: Interpret S224 phosphorylation data within the context of cell cycle phase:

  • S224 phosphorylation occurs in a cell cycle-dependent manner, with highest levels likely during S/G2 phases when CDK2-cyclin A is active

  • Changes in S224 phosphorylation may reflect either altered CDK2 activity or changes in ATRIP protein levels

• Checkpoint Function Correlation: Correlate S224 phosphorylation levels with:

  • CHK1 phosphorylation (a downstream ATR target)

  • G2/M transition rates

  • Recovery from replication stress

  • Cellular sensitivity to genotoxic agents

• Integration with ATR Activity: Consider how S224 phosphorylation correlates with other indicators of ATR pathway activation:

  • RPA32 phosphorylation

  • γH2AX foci formation

  • TOPBP1 recruitment to damage sites

What experimental controls are essential when studying ATRIP S224 phosphorylation?

Essential experimental controls for studying ATRIP S224 phosphorylation include:

• Antibody Validation Controls:

  • Phosphatase-treated samples (negative control)

  • S224A mutant-expressing cells (negative control)

  • S224D/E mutant-expressing cells (positive control surrogate)

  • siRNA-depleted cells (specificity control)

• Cell Cycle Controls:

  • Synchronized cell populations at defined cell cycle stages

  • CDK2 inhibitor-treated cells (roscovitine or similar compounds)

  • Cyclin A co-immunoprecipitation to confirm interaction

• DNA Damage Controls:

  • Dose-response curves for DNA damaging agents

  • Time-course experiments to distinguish initial activation from maintenance

  • ATR inhibitor treatments to determine dependence on ATR kinase activity

• Functional Checkpoint Controls:

  • G2/M transition rate measurements

  • Mitotic index quantification

  • Parallel assessment of checkpoint proteins (CHK1, CDC25C)

How can ATRIP S224 phosphorylation research contribute to understanding cancer biology and therapeutics?

ATRIP S224 phosphorylation research has significant implications for cancer biology and therapeutics:

• Checkpoint Dysregulation in Cancer:

  • Cancer cells often exhibit altered cell cycle checkpoint function

  • Analyzing S224 phosphorylation patterns in tumor samples versus normal tissues may reveal specific checkpoint defects

  • The ATR pathway represents a therapeutic vulnerability in cancers with defective G1 checkpoints

• Therapeutic Targeting Strategies:

  • Combination therapies targeting both CDK2 and ATR pathways may show synergistic effects

  • S224 phosphorylation status could serve as a biomarker for response to checkpoint inhibitors

  • Developing compounds that specifically disrupt the CDK2-ATRIP interaction might offer selective targeting of checkpoint maintenance

• Resistance Mechanisms:

  • Alterations in ATRIP S224 phosphorylation might contribute to resistance against:

    • Radiation therapy

    • Chemotherapeutics causing replication stress

    • PARP inhibitors

    • ATR/CHK1 inhibitors

• Predictive Biomarker Development:

  • Phospho-ATRIP (S224) antibodies could be developed as diagnostic tools to:

    • Predict therapy responses

    • Monitor treatment efficacy

    • Identify patients likely to benefit from specific combination regimens