Phospho-TP53 (Ser366) Antibody

What is p53/TP53 and what roles does it play in cellular function?

p53 (also known as TP53) is a crucial tumor suppressor protein that functions as a sequence-specific transcription factor activated by cellular stress. It mediates cell cycle arrest or apoptosis in response to DNA damage or nucleotide starvation. Structurally, p53 comprises four main domains: an N-terminal transactivation domain, a central DNA-binding domain, an oligomerization domain, and a C-terminal regulatory domain. In response to stress signals, p53 is upregulated and activated to induce transcription of specific genes involved in G1 or G2/M arrest, including p21waf1. Under normal conditions, p53 is maintained at low levels in cells, but its expression increases significantly in transformed cell lines, contributing to transformation and malignancy. Notably, TP53 is the most commonly mutated cancer driver gene, with mutations occurring both as somatic alterations in human malignancies and as germline mutations in cancer-prone families such as those with Li-Fraumeni syndrome .

Why is phosphorylation at Serine 366 significant in p53 function?

Phosphorylation at Serine 366 represents a key post-translational modification that regulates p53 activity in response to DNA damage. Research has demonstrated that Ser366 is phosphorylated by checkpoint kinase 1 (CHK1) in response to treatments like camptothecin, a topoisomerase inhibitor. This phosphorylation event is functionally important, as reduced phosphorylation at Ser366 correlates with decreased expression of apoptosis-related genes like BAX. When CHK1 is silenced in cancer cell lines like LNCaP, both Ser366 phosphorylation and apoptotic response are markedly diminished, suggesting this modification plays a critical role in the p53-mediated apoptotic pathway. Unlike some other phosphorylation sites that primarily affect protein stability, Ser366 phosphorylation appears more directly involved in determining the transcriptional output of p53, particularly regarding pro-apoptotic gene expression .

How does Phospho-TP53 (Ser366) Antibody specifically detect this modification?

Phospho-TP53 (Ser366) antibodies are engineered to recognize the unique conformational epitope created when serine at position 366 is phosphorylated. These antibodies demonstrate high specificity for the phosphorylated form versus the non-phosphorylated peptide, as confirmed through ELISA validation studies. The specificity is achieved through careful immunogen design, typically using synthesized phospho-peptides that mimic the region around phosphorylated Ser366. The antibodies are generally affinity-purified from rabbit antiserum using epitope-specific immunogen chromatography to ensure high specificity. This allows researchers to detect endogenous levels of p53 only when phosphorylated at this specific residue, even in complex cellular lysates. Validation studies show clear discrimination between phosphorylated and non-phosphorylated forms, with the antibody typically showing minimal cross-reactivity with other phosphorylation sites on p53 or with phosphorylated residues on other proteins .

What applications is the Phospho-TP53 (Ser366) antibody suitable for?

The Phospho-TP53 (Ser366) antibody has been validated for several key research applications. Western blotting represents the primary application, with recommended dilution ranges of 1:500-2000 for optimal results. ELISA applications are also well-established, with higher dilution ratios (1:10000-20000) typically recommended for this more sensitive technique. Cell-based ELISA formats allow for detection of phosphorylated p53 in fixed cells, enabling analysis of phosphorylation events in their cellular context. Some antibody preparations may also be suitable for immunoprecipitation, though this should be validated for each specific antibody product. Importantly, these antibodies are strictly for research use only and must not be used in diagnostic or therapeutic applications. The reactivity profile typically includes human, rat, and mouse samples, making these antibodies versatile tools for comparative studies across mammalian model systems .

What methodology should be followed for Western Blot detection of phosphorylated p53 at Ser366?

For optimal Western Blot detection of phosphorylated p53 at Ser366, researchers should follow these methodological guidelines:

• Sample preparation: Extract proteins from cells using lysis buffers containing phosphatase inhibitors to prevent dephosphorylation during processing. UV treatment (5 minutes) of cells prior to lysis can enhance Ser366 phosphorylation for positive controls.

• Gel electrophoresis: Use 10-12% SDS-PAGE gels for optimal resolution around the 53 kDa mark where p53 migrates.

• Transfer and blocking: After transfer to PVDF or nitrocellulose membranes, block with 5% BSA (preferred over milk for phospho-epitopes) in TBST buffer.

• Antibody incubation: Dilute Phospho-p53 (Ser366) antibody 1:500-2000 in blocking buffer and incubate overnight at 4°C with gentle agitation.

• Detection controls: Include extracts from untreated cells as negative controls and UV-treated or camptothecin-treated samples as positive controls.

• Validation: Always run a total p53 antibody on parallel samples to normalize phospho-signal to total protein levels.

• Stripping and reprobing: If verification with multiple antibodies is required, gentle stripping buffers should be used to avoid epitope damage.

When interpreting results, the phosphorylated p53 should appear as a single band at approximately 53 kDa, and this signal should increase following appropriate stimuli like DNA damage .

What are the critical parameters for successful cell-based ELISA using Phospho-TP53 (Ser366) antibody?

For cell-based ELISA applications with Phospho-TP53 (Ser366) antibody, several critical parameters must be controlled:

• Cell line selection: The cell line must express detectable levels of p53 protein. The assay can detect phospho-p53 expression in as few as 5,000 HeLa cells when properly optimized.

• Cell density: Cells should be 75-90% confluent at the time of treatment. For adherent cells like HeLa, seeding approximately 30,000 cells per well in a 96-well plate overnight is typically optimal.

• Substrate preparation: For suspension cells or loosely attached cells, pre-coating plates with 100 μl of 10 μg/ml Poly-L-Lysine for 30 minutes at 37°C improves adherence.

• Fixation protocol: Use 8% formaldehyde for suspension cells to ensure proper attachment and preservation of the phospho-epitope.

• Antibody concentration: Follow the manufacturer's recommended dilution ratios, typically much higher (1:10000-20000) for ELISA than for Western blotting due to the higher sensitivity of ELISA detection systems.

• Controls: Include wells treated with phosphorylation-inducing agents (such as UV or camptothecin) as positive controls, and samples treated with phosphatase as negative controls.

• Normalization: Normalize phospho-p53 signal to total p53 levels or to housekeeping proteins like GAPDH for accurate quantification of phosphorylation status independent of protein expression levels.

Maintaining these parameters ensures reproducible detection of Ser366 phosphorylation across experimental conditions .

How does Ser366 phosphorylation of p53 relate to DNA damage response and cancer?

Serine 366 phosphorylation represents a critical regulatory event in the p53-mediated DNA damage response pathway. Upon DNA damage, checkpoint kinase 1 (CHK1) phosphorylates p53 at Ser366 and Thr387, which serves as a molecular switch directing cellular fate toward apoptosis. This phosphorylation event is particularly important for activating pro-apoptotic genes like BAX. In cancer research, the status of Ser366 phosphorylation can provide valuable insights into tumor response to genotoxic therapies and potential resistance mechanisms. Notably, when CHK1 is silenced in cancer cells like LNCaP, both Ser366 phosphorylation and apoptotic response to DNA damage agents are significantly reduced, suggesting that defects in this phosphorylation pathway could contribute to therapy resistance. Unlike some other p53 phosphorylation events that primarily affect protein stability, Ser366 phosphorylation appears more directly involved in determining the transcriptional output of p53, particularly regarding apoptotic gene expression patterns. Understanding this phosphorylation event may therefore provide opportunities for developing more targeted cancer therapies .

What is known about the kinases responsible for Ser366 phosphorylation?

Checkpoint kinase 1 (CHK1) has been identified as the primary kinase responsible for phosphorylating p53 at Serine 366 in response to DNA damage. Research in LNCaP cells treated with camptothecin demonstrated that CHK1 simultaneously phosphorylates Ser366 and Thr387 on p53. This dual phosphorylation appears to be functionally significant, as silencing CHK1 leads to reduced phosphorylation at both sites and decreased expression of apoptosis-related genes. Unlike some other p53 phosphorylation sites that are targeted by multiple kinases, current evidence suggests that Ser366 is primarily regulated by CHK1, making it a relatively specific readout of CHK1 activity in cells. This specificity may be valuable for researchers studying CHK1 signaling pathways or developing CHK1-targeted therapies. The CHK1-p53 Ser366 phosphorylation axis represents a distinct signaling mode in the DNA damage response network, particularly connecting checkpoint activation to apoptotic outcomes in damaged cells .

How does phosphorylation at Ser366 compare with other p53 phosphorylation sites?

Phosphorylation of p53 occurs at multiple sites throughout the protein, with each modification having distinct regulatory implications:

• N-terminal phosphorylation (e.g., Ser15, Ser20): Primarily affects p53 stability by preventing MDM2-mediated degradation, representing the first phase of p53 activation.

• Central domain phosphorylation: Less common but can affect DNA binding properties.

• C-terminal phosphorylation (including Ser366): Often modulates transcriptional activity rather than stability.

Ser366 phosphorylation differs from other well-characterized sites in several ways:

• Unlike Ser15 phosphorylation, which is primarily associated with protein stabilization, Ser366 phosphorylation appears more directly linked to transcriptional activation of specific gene sets, particularly apoptotic genes.

• While Ser46 phosphorylation is considered a major determinant for apoptosis induction, Ser366 works in concert with other C-terminal modifications (like Thr387) to fine-tune this response.

• Ser392 phosphorylation (by CK2) affects G2 arrest, whereas Ser366 phosphorylation (by CHK1) appears more specifically connected to apoptotic outcomes.

The distinct patterning of these phosphorylation events likely serves as a molecular code that determines the ultimate cellular response to stress stimuli - whether cell cycle arrest, DNA repair, senescence, or apoptosis .

How can researchers address the challenge of detecting low-abundance phosphorylation events like Ser366?

Detecting low-abundance phosphorylation events such as p53 Ser366 phosphorylation presents significant technical challenges. Researchers can implement several strategies to enhance detection sensitivity:

• Phospho-enrichment techniques: Prior to analysis, use phospho-peptide enrichment methods such as titanium dioxide (TiO₂) or immobilized metal affinity chromatography (IMAC) to concentrate phosphorylated proteins.

• Inducing phosphorylation: Treat cells with appropriate stimuli known to induce Ser366 phosphorylation, such as camptothecin or UV radiation, to increase the abundance of the target modification.

• Phosphatase inhibition: Include comprehensive phosphatase inhibitor cocktails in all lysis and sample preparation buffers to prevent dephosphorylation during processing.

• Signal amplification methods: For Western blotting, consider using high-sensitivity ECL substrates or fluorescent detection systems with digital imaging rather than film.

• Alternative techniques: When protein quantities are limiting, consider using ELISA-based methods which typically offer greater sensitivity than Western blotting, with detection possible from as few as 5,000 cells.

• Quantitative mass spectrometry: For precise quantification of phosphorylation stoichiometry, consider targeted mass spectrometry approaches using heavy-labeled synthetic phosphopeptide standards.

• Antibody validation: Rigorously validate antibody specificity using phosphopeptide competition assays and phosphatase-treated negative controls to ensure signal authenticity.

These approaches can significantly improve detection of low-abundance phosphorylation events, enabling more reliable analysis of p53 post-translational modifications under various experimental conditions .

What experimental controls are essential when studying p53 Ser366 phosphorylation?

When investigating p53 Ser366 phosphorylation, implementing proper controls is critical for result interpretation and validation:

• Positive controls:

  • Cells treated with known inducers of Ser366 phosphorylation (e.g., camptothecin, UV exposure for 5 minutes)

  • Recombinant phosphorylated peptides corresponding to the Ser366 region

  • Cells overexpressing CHK1, which increases Ser366 phosphorylation

• Negative controls:

  • Phosphatase-treated samples to demonstrate phosphorylation specificity

  • CHK1-silenced or inhibitor-treated cells showing reduced Ser366 phosphorylation

  • Cells expressing p53 with S366A mutation to confirm antibody specificity

• Specificity controls:

  • Peptide competition assays using phosphorylated versus non-phosphorylated peptides

  • Comparative analysis with other p53 phospho-specific antibodies

  • Parallel blots probed with total p53 antibody to normalize phospho-signal

• Loading controls:

  • GAPDH or other housekeeping proteins to ensure equal loading

  • Total p53 detection to distinguish changes in phosphorylation from changes in protein abundance

• Biological validation:

  • Correlation of phosphorylation status with expected downstream effects (e.g., apoptosis markers, BAX expression)

  • Replication across multiple cell lines to ensure observations aren't cell-type specific

These controls collectively ensure that observed changes in Ser366 phosphorylation are biological rather than technical in nature and accurately reflect the phenomenon being studied .

How can researchers distinguish between effects of multiple phosphorylation events on p53?

Distinguishing between the effects of multiple phosphorylation events on p53 requires sophisticated experimental approaches:

• Site-directed mutagenesis: Generate p53 constructs with serine-to-alanine mutations at specific sites (e.g., S366A, S15A) to prevent phosphorylation at individual residues, allowing assessment of each site's contribution to p53 function.

• Phospho-specific antibody arrays: Use antibody panels recognizing different phosphorylation sites to simultaneously monitor multiple modifications across various conditions.

• Mass spectrometry-based phosphoproteomics: Implement quantitative MS approaches to simultaneously measure phosphorylation stoichiometry at multiple sites, revealing temporal dynamics and correlations between different phosphorylation events.

• Kinase inhibition strategies: Selectively inhibit specific kinases (e.g., CHK1 for Ser366, ATM for Ser15) to dissect kinase-specific phosphorylation patterns and their consequences.

• Phosphomimetic mutants: Create S366D or S366E mutations to mimic constitutive phosphorylation at specific sites, allowing assessment of phosphorylation effects independent of upstream signaling.

• Temporal analysis: Perform detailed time-course experiments following DNA damage to establish the sequence of phosphorylation events and their relationship to specific cellular outcomes.

• Bioinformatic modeling: Employ computational approaches to model the complex interplay between multiple phosphorylation events and predict their combined effects on p53 conformation and function.

These approaches can help researchers decipher the complex "phosphorylation code" that determines the specific functional outcomes of p53 activation in different cellular contexts .

What are the latest methodological advances in studying p53 post-translational modifications?

Recent methodological advances have significantly enhanced researchers' ability to study p53 post-translational modifications:

• Proximity ligation assays (PLA): Allow visualization of phosphorylated p53 in situ with single-molecule sensitivity, providing spatial information about where in the cell specific modifications occur.

• CRISPR-Cas9 knock-in strategies: Enable introduction of tagged versions of p53 or single-amino-acid mutations at endogenous loci, preserving native expression levels and regulatory mechanisms.

• Single-cell phosphoproteomics: Emerging technologies permit analysis of p53 phosphorylation status at the single-cell level, revealing heterogeneity within populations.

• Targeted proteomics using parallel reaction monitoring (PRM): Allows highly sensitive and specific quantification of multiple p53 phosphorylation sites simultaneously from limited sample amounts.

• Bispecific antibody approaches: Similar to those used in immunotherapy, these can be adapted to recognize specific mutant p53 forms or post-translationally modified variants with increased specificity and sensitivity.

• Structure-based studies: Cryo-EM and crystallography of phosphorylated p53 in complex with interacting proteins provide mechanistic insights into how modifications alter protein function.

• Optogenetic control of kinase activity: Allows precise temporal control of phosphorylation events to dissect kinetics and order-of-operations in phosphorylation cascades affecting p53.

• Digital ELISA technologies: Ultra-sensitive detection methods can measure low-abundance phosphorylated forms of p53 that would be undetectable by conventional approaches.

These advanced methodologies are transforming our understanding of how p53 post-translational modifications, including Ser366 phosphorylation, coordinate complex cellular responses to stress and DNA damage .

What cell line models are most appropriate for studying p53 Ser366 phosphorylation?

Selecting appropriate cell models is crucial for effective study of p53 Ser366 phosphorylation:

• Recommended cell lines:

  • HeLa cells: Well-characterized for p53 phosphorylation studies, with optimal cell density of 30,000 cells per well for 96-well plate assays.

  • LNCaP cells: Documented to display robust Ser366 phosphorylation in response to camptothecin treatment and CHK1 activation.

  • COS7 cells: Shown to exhibit increased p53 phosphorylation following UV treatment, useful for positive control preparations.

  • HCT116 cells: p53 wild-type colorectal cancer cells that maintain intact DNA damage response pathways.

• Cell line considerations:

  • Use cells with wild-type p53 for phosphorylation studies to avoid complications from mutant p53 behavior.

  • Consider p53-null cells (like HCT116 p53-/- paired with p53-positive counterparts) for manipulation and reintroduction of modified p53 constructs.

  • Cell confluence should be maintained at 75-90% for optimal detection of phosphorylation events.

• Specialized models:

  • Neural cell lines (e.g., SH-SY5Y) may show tissue-specific regulation patterns different from epithelial models.

  • Primary cells may exhibit different phosphorylation kinetics than immortalized lines and more accurately represent in vivo responses.

The appropriate cell model should be selected based on the specific research question, considering factors such as p53 status, intact DNA damage response pathways, and tissue relevance to the biological process being studied .

What are the optimal conditions for inducing and detecting p53 Ser366 phosphorylation?

Optimizing conditions for p53 Ser366 phosphorylation requires careful consideration of induction stimuli, timing, and detection methods:

• Effective induction methods:

  • Camptothecin treatment: A topoisomerase I inhibitor demonstrated to induce CHK1-mediated phosphorylation of Ser366.

  • UV irradiation: Short exposure (5 minutes) effectively triggers phosphorylation cascades activating p53.

  • Other DNA-damaging agents: Etoposide, doxorubicin, and ionizing radiation can induce various patterns of p53 phosphorylation.

• Timing considerations:

  • Early phosphorylation events typically occur within 30 minutes to 2 hours post-treatment.

  • Ser366 phosphorylation may show different kinetics than other sites (like Ser15), necessitating time-course experiments.

  • Coordinate sampling with expected downstream effects (e.g., apoptotic gene expression peaks several hours after phosphorylation).

• Buffer and lysate preparation:

  • Include comprehensive phosphatase inhibitor cocktails in all buffers.

  • Maintain cold chain throughout sample processing to minimize dephosphorylation.

  • Rapid processing is essential as phosphorylation marks can be transient.

• Detection optimization:

  • Enrich phosphoproteins when possible to enhance detection sensitivity.

  • For Western blotting, BSA is preferred over milk for blocking when detecting phospho-epitopes.

  • For ELISA applications, optimize antibody concentration through preliminary titration experiments.

The ideal experimental design includes both time-course and dose-response elements to fully characterize Ser366 phosphorylation under specific stimulus conditions .

What quantification methods provide the most reliable measures of p53 Ser366 phosphorylation?

Selecting appropriate quantification methods is essential for reliable measurement of p53 Ser366 phosphorylation:

• Western blot quantification:

  • Use digital imaging systems rather than film for greater dynamic range.

  • Implement ratiometric analysis of phospho-p53 to total p53 to control for expression differences.

  • Include calibration standards of known phosphopeptide concentrations when possible.

• ELISA-based quantification:

  • Cell-based ELISA provides direct measurement in fixed cells without extraction requirements.

  • Sandwich ELISA using capture antibodies for total p53 and detection antibodies for phospho-Ser366 offers high specificity.

  • Standard curves with recombinant phosphorylated proteins enable absolute quantification.

• Mass spectrometry approaches:

  • Selected reaction monitoring (SRM) or parallel reaction monitoring (PRM) provide precise quantification of specific phosphopeptides.

  • AQUA peptides (isotopically labeled reference peptides) enable absolute quantification of phosphorylation stoichiometry.

  • Data-independent acquisition methods offer broader coverage of multiple modification sites simultaneously.

• Immunofluorescence quantification:

  • High-content imaging allows single-cell resolution of phosphorylation status across populations.

  • Z-stack confocal microscopy provides spatial information about phosphorylated p53 localization.

  • Automated image analysis platforms ensure objective quantification across large datasets.

• Flow cytometry:

  • Phospho-flow techniques enable rapid quantification across large cell populations.

  • Multiparameter analysis allows correlation with cell cycle phase or other cellular states.

The optimal quantification approach depends on the specific experimental question, required sensitivity, and available sample material. Combining multiple methodologies provides the most robust confirmation of phosphorylation status .

How should researchers interpret changes in p53 Ser366 phosphorylation in relation to biological outcomes?

Interpreting changes in p53 Ser366 phosphorylation requires careful consideration of multiple factors:

• Contextual analysis framework:

  • Relate phosphorylation changes to concurrent cellular phenotypes (apoptosis, cell cycle arrest, senescence).

  • Analyze gene expression changes of p53 targets, particularly apoptosis regulators like BAX, following Ser366 phosphorylation.

  • Consider the cellular stress context—different stressors may induce distinct phosphorylation patterns with different functional consequences.

• Integrated analysis strategies:

  • Examine Ser366 phosphorylation in relation to other p53 modifications that occur simultaneously.

  • Correlate phosphorylation with p53 subcellular localization and DNA binding activity.

  • Assess changes in protein-protein interactions following phosphorylation to identify effector pathways.

• Temporal considerations:

  • Early phosphorylation events may indicate pathway activation, while sustained phosphorylation suggests maintained signaling.

  • The sequence of phosphorylation at different sites often determines the ultimate biological outcome.

  • Delayed dephosphorylation may indicate dysregulation of phosphatase activity.

• Quantitative thresholds:

  • Determine whether phosphorylation operates in a threshold-dependent or graded manner for downstream effects.

  • Establish dose-response relationships between stimulus intensity, phosphorylation levels, and biological outcomes.

• Cell-type variations:

  • Consider that the same phosphorylation event may have different consequences in different cellular contexts.

  • Compare observations across multiple cell types to identify consistent versus context-specific effects.

Meaningful interpretation requires integrating phosphorylation data with other molecular and phenotypic measurements to establish causal relationships rather than mere correlations .

What statistical approaches are most appropriate for analyzing phosphorylation data across experimental conditions?

• Normalization strategies:

  • Normalize phospho-signal to total protein expression to account for expression level differences.

  • Consider using multiple housekeeping proteins or global protein normalization for Western blot data.

  • For mass spectrometry data, implement appropriate normalization for run-to-run variation.

• Statistical testing frameworks:

  • For comparing two conditions: paired t-tests for before/after treatments in the same cells.

  • For multiple conditions: ANOVA with appropriate post-hoc tests (Tukey, Dunnett) to control for multiple comparisons.

  • For non-normally distributed data: non-parametric alternatives like Mann-Whitney U or Kruskal-Wallis tests.

• Dose and time-response analyses:

  • Fit appropriate pharmacological models (e.g., sigmoidal dose-response) to determine EC50 values for phosphorylation induction.

  • Time-course data may require more complex modeling approaches like impulse models or oscillation analysis.

• Multivariate approaches:

  • Principal component analysis (PCA) or partial least squares discriminant analysis (PLS-DA) for data with multiple phosphorylation sites measured simultaneously.

  • Hierarchical clustering to identify patterns of phosphorylation across conditions.

  • Correlation networks to visualize relationships between different phosphorylation events.

• Power analysis:

  • Calculate required sample sizes based on expected effect sizes and variability to ensure adequate statistical power.

  • Consider biological replicates (different experiments) versus technical replicates (repeated measurements).

• Reporting standards:

  • Always report both statistical significance (p-values) and effect sizes with confidence intervals.

  • Include detailed information about normalization procedures, outlier handling, and statistical tests performed.

These approaches ensure robust analysis of phosphorylation data while avoiding common statistical pitfalls in signal interpretation .

How can researchers distinguish between direct effects of Ser366 phosphorylation and secondary consequences?

Distinguishing direct effects of Ser366 phosphorylation from secondary consequences requires strategic experimental approaches:

• Site-specific mutational analysis:

  • Generate S366A (phospho-null) mutants that cannot be phosphorylated to identify functions requiring this modification.

  • Create S366D/E (phosphomimetic) mutants to simulate constitutive phosphorylation independent of upstream signaling.

  • Compare phenotypes of these mutants to determine direct consequences of Ser366 phosphorylation.

• Temporal resolution studies:

  • Implement high-resolution time-course studies to establish cause-effect relationships.

  • Early events following phosphorylation are more likely direct consequences.

  • Use rapid kinase inhibition to acutely block phosphorylation and identify immediate effects.

• Proteomic approaches:

  • Identify direct binding partners that specifically recognize phosphorylated Ser366 using phospho-specific pull-downs.

  • Compare interactomes of wild-type versus phosphomimetic/phospho-null mutants to identify phosphorylation-dependent interactions.

  • Employ proximity labeling techniques to identify proteins in close proximity to phosphorylated p53.

• Transcriptional profiling:

  • Conduct RNA-seq at early timepoints after phosphorylation to identify immediate transcriptional responses.

  • Compare transcriptional profiles of phosphomimetic versus phospho-null mutants to identify phosphorylation-dependent gene expression.

• Pathway inhibition strategies:

  • Systematically inhibit downstream pathways to determine which effects of Ser366 phosphorylation depend on specific effector pathways.

  • Use combinatorial approaches to dissect complex signaling networks downstream of phosphorylation.

• In vitro biochemical assays:

  • Test direct effects of phosphorylation on p53 properties like DNA binding affinity, oligomerization, or interaction with cofactors.

  • Use recombinant proteins with site-specific phosphorylation to eliminate cellular complexity.