Phospho-TP63 (S395) Antibody

What is TP63 and what is the significance of its phosphorylation at S395?

TP63 (tumor protein 63) is a transcription factor belonging to the p53 family with critical roles in development, cellular differentiation, and stress response. Phosphorylation at serine 395 (S395) represents a key post-translational modification that regulates p63 activity. Research indicates that S395 phosphorylation induces the SAPK/JNK signaling pathway and triggers apoptosis in oocytes and granulosa cells . This modification appears particularly relevant during DNA damage responses, where phosphorylated p63 can trigger multiple death receptor pathways including CD95, TNF-R, and TRAIL-R cell death-related NF-κB pathways, consequently sensitizing cells toward apoptosis .

What are the main isoforms of TP63 and how do they differ functionally?

TP63 exists in multiple isoforms arising from alternative promoter usage and differential splicing:

Research demonstrates that ΔNp63α is the most abundant isoform in human airway epithelial basal cells, while TAp63 isoforms are expressed at much lower levels in these cells . Functionally, these isoforms exhibit distinct and sometimes opposing properties:

• TA-p63α cannot drive transcription on the p53-responsive element PG13, unlike TA-p63β and TA-p63γ

• The α isoform inhibits epithelial-to-mesenchymal transition (EMT) and regulates apoptosis

• The γ isoform promotes EMT and correlates with poor prognosis in head and neck squamous cell carcinoma

These differential activities stem partly from structural variations, including the presence of an inhibitory "transactivation inhibitory domain" (TID) in the α-specific C-terminal region .

What are the optimal methods for detecting TP63 phosphorylation at S395?

Several validated methodologies exist for detecting TP63 phosphorylation at S395:

• Western Blotting (WB):

  • Recommended dilutions: 1:500-1:2000

  • Validated cell lines: LOVO, A549, and HepG2 cells treated with nocodazole (1μg/ml for 18h)

  • Critical control: Run parallel blots with phospho-peptide blocking to confirm specificity

• Cell-Based ELISA:

  • Can detect phospho-p63 in as few as 5,000 cells

  • Normalization options:

    • Total p63 normalization: Calculate OD450(Phospho-S395)/OD450(Total p63)

    • GAPDH normalization: Normalize using housekeeping protein expression

    • Crystal violet staining: Calculate OD450/OD595 ratio

• Immunohistochemistry/Immunofluorescence:

  • Particularly useful for analyzing patient samples and spatial distribution

  • Validated in human, mouse, and rat tissues

The selection of technique depends on your specific research question, sample availability, and required quantification accuracy.

How should I validate the specificity of a Phospho-TP63 (S395) antibody?

Rigorous validation is essential for ensuring reliable results with phospho-specific antibodies:

• Phospho-peptide competition assay: Western blot analysis with and without blocking using the specific phospho-peptide. The signal should be eliminated when blocked with the phospho-peptide if the antibody is truly phospho-specific .

• Phosphatase treatment control: Samples treated with phosphatase should show diminished signal with a phospho-specific antibody while maintaining total protein signal.

• siRNA-mediated knockdown: Using siRNAs targeting p63 should proportionally reduce phospho-specific signal .

• Positive and negative controls:

  • Positive: Cells treated with known inducers of S395 phosphorylation (e.g., nocodazole)

  • Negative: Untreated cells or cells where phosphorylation is inhibited

• Multiple detection methods: Confirm findings using complementary techniques such as mass spectrometry.

Proper validation ensures that experimental results truly reflect the phosphorylation status of TP63 at S395 rather than non-specific binding or artifacts.

What is the detailed protocol for using Phospho-TP63 (S395) antibody in cell-based ELISA assays?

Based on established colorimetric cell-based ELISA protocols :

• Cell preparation:

  • For adherent cells: Seed 30,000 cells/well in 96-well plates (75-90% confluence)

  • For suspension cells: Pre-coat wells with 10μg/ml Poly-L-Lysine for 30 minutes at 37°C

• Treatment application (if studying induction):

  • Apply inhibitors, activators, or stressors according to experimental design

• Fixation and processing:

  • Add 100μl Fixing Solution, incubate 20 minutes at room temperature

  • Add 100μl Quenching Buffer, incubate 20 minutes at room temperature

  • Add 200μl Blocking Buffer, incubate 1 hour at room temperature

• Antibody incubations:

  • Primary antibodies (50μl): Anti-p63 (Phospho-Ser395), Anti-total p63, Anti-GAPDH

  • Incubate 16 hours at 4°C (or 2 hours at room temperature for high-expression targets)

  • Secondary antibodies (50μl): HRP-conjugated, incubate 1.5 hours at room temperature

• Detection:

  • Add 50μl substrate, incubate 30 minutes at room temperature

  • Add 50μl stop solution, read absorbance at 450nm

• Data normalization:

  • Calculate ratios of phospho-p63 to total p63 or GAPDH

  • Alternatively, normalize to cell number using crystal violet staining (OD595)

This protocol enables quantitative measurement of p63 phosphorylation status while preserving cellular context, offering advantages over homogenized sample-based methods.

How does TP63 phosphorylation at S395 mechanistically regulate DNA damage response and apoptosis?

TP63 phosphorylation at S395 serves as a molecular switch in the DNA damage response cascade:

• Stabilization mechanism: Under normal conditions, TAp63 proteins undergo rapid degradation via the ubiquitin-proteasome pathway . Following genotoxic stress (UV irradiation, actinomycin D, bleomycin, etoposide), TAp63 protein levels increase .

• Protein-protein interactions: The interaction between phosphorylated TAp63 and Cables1 stabilizes the TAp63 isoform structure, preventing degradation .

• Signaling pathway activation: Phosphorylated p63 (S395) activates the SAPK/JNK signaling pathway, a key mediator of stress-induced apoptosis .

• Death receptor upregulation: TAp63 stimulation triggers upregulation of multiple death receptors (CD95, TNF-R, TRAIL-R) within the NF-κB pathway, increasing cellular sensitivity to apoptotic signals .

This integrated mechanism ensures that cells with DNA damage either repair the damage or undergo programmed cell death, preventing potential malignant transformation. The phosphorylation event thus serves as a critical quality control checkpoint in cellular stress response.

What is the relationship between TP63 isoforms, phosphorylation status, and cancer progression?

The relationship between TP63 isoforms, their phosphorylation, and cancer progression reveals complex, context-dependent patterns:

• Expression patterns and clinical outcomes:

  • TP63 is frequently amplified or overexpressed in squamous cell carcinomas

  • Higher TP63 expression correlates with recurrence in esophageal squamous cell carcinoma (ESCC)

  • Patients with strong TP63 expression show poorer prognosis and lower 3-year recurrence-free survival rates

• Isoform-specific effects:

  • TP63γ isoform proportion serves as a detrimental factor for survival in head and neck squamous cell carcinoma (HNSCC) patients

  • TP63γ expression correlates with downregulation of desmosomal genes, potentially facilitating metastasis

  • The α isoform inhibits EMT while the γ isoform promotes it, suggesting divergent roles in tumor progression

• Regulatory mechanisms:

  • The splicing factor PTBP1 represses TP63γ isoform production by directly binding to TP63 pre-mRNA near the TP63γ-specific exon

  • This regulatory mechanism represents a potential therapeutic target for controlling isoform ratios

These findings suggest that quantifying specific TP63 isoforms and their phosphorylation status could serve as biomarkers for cancer progression and treatment response. The balance between different isoforms, rather than absolute expression levels, may be more informative for prognostic purposes.

How can I design experiments to differentiate between the activities of different TP63 isoforms?

To differentiate between TP63 isoform-specific activities, consider this comprehensive experimental approach:

• Isoform-specific expression analysis:

  • Quantitative RT-PCR with isoform-specific primers

  • Western blotting with antibodies that distinguish between isoforms

  • RNA-seq analysis with isoform-specific mapping

• Overexpression systems:

  • Adenoviral vectors encoding specific isoforms (ΔNp63α, TAp63α, TAp63γ)

  • Optimize infection parameters based on cell type:

    • Primary epithelial cells: MOI 0.15 pfu/cell

    • A549 cells: MOI 1.0-3.0 pfu/cell

  • Collect RNA at 48 hours and protein at 72 hours post-infection

• Selective knockdown approaches:

  • Design siRNAs targeting specific domains of p63

  • Validate knockdown specificity using isoform-specific primers/antibodies

• Functional readouts:

  • Transcriptional activity: Reporter assays with promoters differentially regulated by specific isoforms

  • Wound repair assays: Quantify repair at 0, 4, 8, and 24 hours post-wounding

  • Epithelial gene expression: Custom arrays containing primers for potential p63-regulated genes

  • EMT marker analysis: Distinguish between α isoforms (inhibit EMT) and γ isoforms (promote EMT)

• Phosphorylation-specific analysis:

  • Compare phosphorylation patterns across isoforms using phospho-specific antibodies

  • Create phospho-mimetic (S395D/E) and phospho-deficient (S395A) mutants to isolate effects

This multi-faceted approach provides complementary data points to comprehensively characterize isoform-specific activities in your experimental system.

What techniques can evaluate the functional consequences of TP63 S395 phosphorylation?

To investigate the functional impact of S395 phosphorylation, consider these methodological approaches:

• Phosphorylation state manipulation:

  • Phospho-mimetic mutants: Replace S395 with glutamic acid (S395E) or aspartic acid (S395D)

  • Phospho-deficient mutants: Replace S395 with alanine (S395A)

  • Kinase inhibitor treatment: Target kinases responsible for S395 phosphorylation

• Gene expression profiling:

  • RT2 Profiler Arrays targeting p63-regulated genes

  • RNA-seq comparing wild-type vs. phospho-mutant expressing cells

  • ChIP-seq to identify differential genomic binding sites

• Protein interaction studies:

  • Co-immunoprecipitation to identify phosphorylation-dependent protein interactions

  • Proximity ligation assays to visualize interactions in situ

  • Mass spectrometry to quantify phosphorylation-dependent binding partners

• Cellular phenotype assays:

  • Apoptosis quantification: Given S395 phosphorylation's role in apoptosis induction

  • Cell cycle analysis: Flow cytometry to assess cell cycle distribution

  • Migration/invasion assays: Transwell or wound healing assays

• Signaling pathway analysis:

  • SAPK/JNK pathway activation: Measure phosphorylation of c-Jun and other downstream targets

  • Death receptor pathways: Analyze CD95, TNF-R, and TRAIL-R expression and activity

By systematically comparing cells expressing wild-type TP63 versus phospho-mutants across these assays, you can comprehensively characterize how S395 phosphorylation alters TP63 function and cellular responses to stress or damage.

Which cell lines and experimental conditions are optimal for studying Phospho-TP63 (S395)?

Selection of appropriate models is critical for studying Phospho-TP63 (S395):

• Validated cell lines:

  • LOVO, A549, and HepG2 cells show detectable phospho-TP63 (S395) following nocodazole treatment

  • Primary human airway epithelial cells (pHAECs) express predominantly the ΔNp63α isoform

  • HBEC6-KT cells are suitable for wound repair studies involving p63

• Induction conditions:

  • Nocodazole treatment (1μg/ml for 18h) effectively induces S395 phosphorylation

  • Other genotoxic agents: UV irradiation, actinomycin D, bleomycin, and etoposide

• Cell culture parameters:

  • For epithelial cells: When submerged in culture, cells form a relatively homogenous cuboidal monolayer representative of basal cells

  • Cell density: 70-90% confluence is optimal for most experiments

  • Transfection conditions: 50nM siRNA with appropriate transfection reagent (e.g., HiPerfect)

• Time course considerations:

  • RNA collection: 48 hours post-treatment/transfection

  • Protein analysis: 72 hours post-treatment/transfection

  • Wound repair assessment: 0, 4, 8, and 24 hours post-wounding

Selecting the appropriate experimental system based on your specific research question will maximize the likelihood of obtaining physiologically relevant results.

How do I accurately normalize and interpret Phospho-TP63 (S395) data in different experimental contexts?

Proper normalization is crucial for meaningful interpretation of phosphorylation data:

• Western blot normalization:

  • Total p63 normalization: Divide phospho-p63 signal by total p63 signal

  • Loading control normalization: Use housekeeping proteins like GAPDH

  • Phospho-peptide competition: Include blocked control to verify specificity

• Cell-based ELISA normalization options:

  • Total p63 normalization: Calculate OD450(Phospho-S395)/OD450(Total p63)

  • GAPDH normalization: Use OD450 values obtained for GAPDH

  • Cell number normalization: Crystal violet staining (OD595) to account for differences in cell density

• Gene expression normalization:

  • Use stable reference genes (e.g., GAPDH) for qRT-PCR data

  • For custom arrays, normalize first to housekeeping genes, then to untreated control values

• Interpretation considerations:

  • Baseline phosphorylation: Establish baseline levels in unstimulated conditions

  • Temporal dynamics: Consider time-dependent changes in phosphorylation status

  • Isoform specificity: Account for different phosphorylation patterns across isoforms

  • Biological relevance: Correlate phosphorylation changes with functional outcomes

• Statistical analysis:

  • Perform experiments in triplicate for statistical robustness

  • Apply appropriate statistical tests based on data distribution

  • Report fold changes relative to controls rather than absolute values

Proper normalization and interpretation enable meaningful comparisons across experimental conditions and between different studies, facilitating broader scientific understanding of TP63 regulation.