Phospho-CDKN2A (Ser152) Antibody

What is CDKN2A/p16INK4a and what is the significance of Ser152 phosphorylation?

CDKN2A encodes multiple proteins, with p16INK4a being a crucial tumor suppressor that inhibits CDK4/CDK6 activity, thereby regulating cell cycle progression. The gene is frequently mutated or deleted in various tumors, functioning as a negative regulator of normal cell proliferation .

Ser152 is a specific phosphorylation site located near the C-terminus of p16INK4a. According to the post-translational modification table in the search results, Ser152 is phosphorylated by ATR (Ataxia Telangiectasia and Rad3-related protein, UniProt ID: Q13535), a key kinase involved in DNA damage response . The position of this phosphorylation site suggests it may play a role in modulating p16INK4a's tumor suppressor function, potentially by affecting protein stability, subcellular localization, or interaction with binding partners.

What are the technical specifications of commercially available Phospho-CDKN2A (Ser152) Antibodies?

Available Phospho-CDKN2A (Ser152) Antibodies are typically rabbit polyclonal antibodies generated against synthetic phosphopeptides derived from human CDKN2A/p16INK4a around the Ser152 phosphorylation site. Key specifications include:

The antibodies are typically purified through affinity chromatography using phospho-peptide and non-phospho-peptide columns to ensure specificity for the phosphorylated form .

How does phosphorylation at Ser152 potentially affect p16INK4a's tumor suppressor function?

Phosphorylation at Ser152 may influence p16INK4a's tumor suppressor activity through several mechanisms:

• CDK4/6 binding modulation: The phosphorylation could alter the protein's conformation, potentially affecting its ability to bind and inhibit CDK4/6 complexes.

• DNA damage response integration: Since ATR (a DNA damage-sensing kinase) phosphorylates Ser152, this modification likely connects DNA damage sensing to cell cycle arrest mechanisms .

• Protein stability regulation: Phosphorylation may alter p16INK4a's susceptibility to degradation, affecting its half-life and concentration within cells.

• Subcellular localization: The added negative charge from phosphorylation could influence p16INK4a's nuclear-cytoplasmic shuttling, impacting its access to CDK targets.

• Protein-protein interactions: Beyond CDK4/6, phosphorylation might modulate interactions with other cellular proteins, affecting additional functions beyond direct CDK inhibition.

Understanding these mechanisms requires experimental approaches that combine phospho-specific antibody detection with functional assays measuring cell cycle progression, protein stability, and protein-protein interactions.

How can researchers use the Phospho-CDKN2A (Ser152) Antibody to investigate functional differences between CDKN2A variants?

CDKN2A variants of uncertain significance (VUSs) present significant challenges in cancer research and clinical genetics. Phospho-CDKN2A (Ser152) Antibody can help elucidate functional differences through several approaches:

• Comparative phosphorylation analysis: Express wild-type and variant p16INK4a in CDKN2A-null cell lines (such as PANC-1, which has homozygous deletion of CDKN2A) , then compare phosphorylation levels using the antibody.

• Structure-function relationships: Determine if variants near Ser152 affect phosphorylation efficiency, providing insights into structural requirements for this modification.

• DNA damage response: Since Ser152 is phosphorylated by ATR, compare how different variants respond to DNA-damaging agents in terms of phosphorylation kinetics.

• Correlation with functional assays: Integrate phosphorylation data with cell proliferation assays (as described in search result ) to determine if phosphorylation status correlates with functional outcomes.

Research has shown that over 40% of CDKN2A VUSs identified in patients with pancreatic ductal adenocarcinoma (PDAC) are functionally deleterious and likely pathogenic . Phosphorylation analysis could provide additional mechanistic insights into variant classification.

What are the recommended protocols for using Phospho-CDKN2A (Ser152) Antibody in Western blotting?

For optimal Western blot detection of phosphorylated p16INK4a at Ser152:

Sample preparation:

• Include phosphatase inhibitors (sodium fluoride, sodium orthovanadate, phosphatase inhibitor cocktails) in lysis buffers

• Maintain samples on ice throughout preparation

• Use protein extraction methods that preserve phospho-epitopes

SDS-PAGE and transfer:

• Use 12-15% gels (p16INK4a is approximately 17kDa)

• Transfer to PVDF membranes at lower voltage for longer time for efficient transfer of smaller proteins

Antibody incubation:

• Block with 5% BSA in TBST (not milk, which contains phosphatases)

• Dilute primary antibody at 1:500-1:3000

• Incubate overnight at 4°C with gentle agitation

Controls:

• Include phosphatase-treated samples as negative controls

• Use CDKN2A knockout cell lysates as specificity controls

• Consider using blocking peptides (such as AF3667-BP) to confirm signal specificity

Detection:

• Use high-sensitivity chemiluminescence detection systems

• Consider stripping and reprobing for total p16INK4a to normalize phosphorylation signal

How can researchers validate the specificity of Phospho-CDKN2A (Ser152) Antibody in their experimental system?

Validating antibody specificity is crucial for reliable experimental results. For Phospho-CDKN2A (Ser152) Antibody, researchers should:

• Peptide competition assay: Pre-incubate the antibody with excess phosphorylated peptide (like AF3667-BP) before application to samples. Specific signals should be eliminated or greatly reduced.

• Phosphatase treatment: Divide samples and treat one portion with lambda phosphatase to remove phosphate groups. The phospho-specific signal should disappear in treated samples.

• Genetic approaches: Use CDKN2A knockout or knockdown models as negative controls. The absence of signal in these models supports antibody specificity.

• Site-directed mutagenesis: Generate expression constructs with Ser152Ala mutations (cannot be phosphorylated) and compare to wild-type. The mutant should show reduced or absent signal.

• Induction experiments: Treat cells with agents known to activate ATR (e.g., UV radiation, replication stress inducers) to increase Ser152 phosphorylation levels and confirm signal enhancement.

• Cross-reactivity assessment: Test the antibody on related phospho-proteins or on samples from species not expected to be recognized to evaluate potential cross-reactivity.

These validation steps ensure that experimental observations genuinely reflect p16INK4a phosphorylation at Ser152.

How can Phospho-CDKN2A (Ser152) Antibody contribute to pancreatic cancer research?

Pathogenic germline CDKN2A variants are associated with increased risk of pancreatic ductal adenocarcinoma (PDAC), with affected individuals having up to a 12.3-fold increased risk . Phospho-CDKN2A (Ser152) Antibody can advance pancreatic cancer research through:

• Variant functional characterization: Functional assays have shown that over 40% of CDKN2A variants of uncertain significance (VUSs) identified in patients with PDAC are functionally deleterious . Phosphorylation analysis can provide additional insights into variant pathogenicity.

• Biomarker development: Investigating whether phosphorylation patterns correlate with disease progression, treatment response, or prognosis in PDAC patients.

• Therapeutic targeting: Identifying kinase-phosphatase networks that regulate p16INK4a phosphorylation could reveal new therapeutic targets.

• Early detection strategies: Since CDKN2A alterations can be early events in pancreatic carcinogenesis, phosphorylation changes might serve as early markers of malignant transformation.

• Genotype-phenotype correlations: Comparing phosphorylation patterns across different CDKN2A variants found in familial pancreatic cancer to understand how specific genetic changes affect protein function.

The International Cancer of the Pancreas Screening (CAPS) Consortium has found that surveillance identified either PDAC or high-grade precursor lesions in 6.8% of high-risk individuals during a median follow-up of 5.6 years . Understanding phosphorylation status could further refine risk assessment models.

What is the relationship between CDKN2A variants, phosphorylation status, and cancer predisposition?

Germline CDKN2A variants that affect p16INK4a have been identified in up to 3.3% of patients with familial pancreatic cancer and up to 2.6% of patients with PDAC unselected for family history . The relationship between these variants, phosphorylation status, and cancer predisposition is complex:

• Structural impacts: Some variants may directly affect the Ser152 residue or nearby regions, potentially altering phosphorylation efficiency by ATR kinase.

• Pathway disruption: Variants that disrupt the DNA damage response-phosphorylation-cell cycle arrest pathway might contribute to genomic instability and cancer development.

• Phosphorylation-dependent functions: If certain p16INK4a functions are phosphorylation-dependent, variants that alter phosphorylation patterns might have selective functional deficits.

• Genetic classification refinement: Incorporating phosphorylation status analysis into variant classification schemes could help reclassify VUSs, which are found in 2.9-4.3% of PDAC patients .

• Personalized surveillance: Understanding how specific variants affect phosphorylation and function could help personalize cancer surveillance protocols for individuals with CDKN2A variants.

A validated in vitro cell proliferation assay found that 12 of 29 germline CDKN2A VUSs previously reported in patients with PDAC were functionally deleterious . Adding phosphorylation analysis could provide mechanistic insights into this functional impairment.

What are the most common technical challenges when working with Phospho-CDKN2A (Ser152) Antibody?

Researchers frequently encounter several challenges when working with phospho-specific antibodies like Phospho-CDKN2A (Ser152) Antibody:

• Low signal intensity: Phosphorylated forms often represent a small fraction of total protein. Solutions include:

  • Enriching phosphorylated proteins through immunoprecipitation

  • Using signal amplification systems

  • Optimizing antibody concentration (try 1:500-1:3000 range for WB, 1:50-1:100 for IHC)

• High background: Polyclonal antibodies may show non-specific binding. Countermeasures include:

  • More stringent blocking (5% BSA in TBST)

  • Increasing wash duration and volume

  • Using affinity-purified antibodies (like those purified via sequential chromatography on phospho-peptide and non-phospho-peptide columns)

• Phosphoepitope instability: Phosphorylations can be lost during sample handling. Prevention strategies include:

  • Adding phosphatase inhibitors immediately upon cell/tissue collection

  • Keeping samples cold throughout processing

  • Avoiding repeated freeze-thaw cycles

• Specificity verification: Confirming phospho-specificity requires appropriate controls:

  • Use blocking peptides like AF3667-BP

  • Include phosphatase-treated samples

  • Test on CDKN2A-null cells like PANC-1

• Tissue-specific expression: p16INK4a is not expressed in all tissues (notably absent in brain and skeletal muscle) , which can lead to false negatives if not considered during experimental design.

Addressing these challenges through careful experimental design and appropriate controls will improve research outcomes when working with this antibody.

What emerging research questions could be addressed using Phospho-CDKN2A (Ser152) Antibody?

Several promising research directions could leverage Phospho-CDKN2A (Ser152) Antibody:

• Phosphorylation dynamics during cell cycle: Investigating how Ser152 phosphorylation changes throughout different cell cycle phases could reveal regulatory mechanisms governing p16INK4a function.

• DNA damage response integration: Since ATR phosphorylates Ser152 , exploring how different types of DNA damage affect this phosphorylation could elucidate p16INK4a's role in genomic stability maintenance.

• Cancer evolution: Analyzing phosphorylation patterns in pre-cancerous lesions versus invasive tumors might reveal whether altered phosphorylation precedes genetic alterations during malignant transformation.

• Therapeutic implications: Determining whether cancer treatments (especially those inducing DNA damage) alter Ser152 phosphorylation could identify potential biomarkers of treatment response.

• Phosphorylation-dependent interactome: Using phospho-specific antibodies to pull down p16INK4a when phosphorylated at Ser152, followed by mass spectrometry, could identify phosphorylation-dependent binding partners.

• Cross-regulation with other phosphorylation sites: p16INK4a contains multiple phosphorylation sites (including S7, S8, Y44, S140, and S152) , and understanding how these sites influence each other could reveal complex regulatory networks.

• Aging and senescence: Given p16INK4a's role in cellular senescence, investigating how Ser152 phosphorylation changes during aging or senescence induction could provide insights into age-related pathologies.

These research directions could significantly advance our understanding of p16INK4a regulation and its implications for cancer biology and therapeutic development.