Phospho-PRKDC (Thr2609) Antibody

What is Phospho-PRKDC (Thr2609) and why is it important in research?

Phospho-PRKDC (Thr2609) refers to the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that is phosphorylated at threonine 2609. This specific phosphorylation is a critical post-translational modification that occurs in response to DNA damage, particularly double-strand breaks (DSBs). The importance of studying this phosphorylation stems from its role in the DNA damage response pathway, specifically in non-homologous end joining (NHEJ), which is the primary repair mechanism for DSBs in mammalian cells.

Research has shown that phosphorylation at Thr2609 is induced rapidly (within minutes) after ionizing radiation and persists for several hours, closely correlating with the timing of DSB repair . Cells expressing a mutant DNA-PKcs where Thr2609 is replaced with alanine (T2609A) show increased radiation sensitivity and impaired ability to repair DSBs, highlighting the functional significance of this phosphorylation event .

How does Phospho-PRKDC (Thr2609) antibody work in detecting DNA damage responses?

Phospho-PRKDC (Thr2609) antibodies are specifically designed to recognize DNA-PKcs only when phosphorylated at threonine 2609. These antibodies are typically generated using synthetic phosphopeptides corresponding to the region surrounding Thr2609 of human DNA-PKcs as immunogens .

When cells experience DNA damage, particularly from ionizing radiation, DNA-PKcs is recruited to DSB sites and undergoes autophosphorylation at several clusters, including the T2609 cluster. The Phospho-PRKDC (Thr2609) antibody binds specifically to this phosphorylated form, allowing researchers to detect and quantify the DNA damage response through various techniques such as Western blotting, immunofluorescence, or immunohistochemistry .

The specificity of these antibodies is crucial and is validated through multiple approaches:

• Using phosphorylated vs. non-phosphorylated peptides

• Testing on wild-type vs. T2609A mutant proteins

• Demonstrating increased signal after DNA damage induction (e.g., ionizing radiation)

• Showing signal reduction after treatment with phosphatase or DNA-PK inhibitors

What experimental techniques can Phospho-PRKDC (Thr2609) antibody be used for?

Phospho-PRKDC (Thr2609) antibodies can be utilized in multiple experimental techniques:

Western Blot (WB): Most commonly used to detect phosphorylated DNA-PKcs as a band around 460 kDa. Typical dilutions range from 1:1000-2000, with nuclear extracts often providing cleaner results than whole cell lysates due to the nuclear localization of DNA-PKcs .

Immunocytochemistry/Immunofluorescence (ICC/IF): Enables visualization of phosphorylated DNA-PKcs in fixed cells, often appearing as distinct nuclear foci at sites of DNA damage. This approach is valuable for studying the kinetics and spatial distribution of DNA-PKcs activation .

Immunohistochemistry (IHC): Used for studying phosphorylated DNA-PKcs in tissue sections, valuable for examining DNA damage responses in vivo or in clinical samples such as cancer tissues or inflammatory conditions .

Immunoprecipitation (IP): For isolating phosphorylated DNA-PKcs from complex protein mixtures for further analysis or to study its interactions with other proteins .

ELISA: For quantitative measurement of phosphorylated DNA-PKcs levels in cell or tissue lysates .

Proximity Ligation Assay (PLA): Advanced technique that allows detection of protein-protein interactions or co-localization of total DNA-PKcs with its phosphorylated form .

What controls should be included when working with Phospho-PRKDC (Thr2609) antibody?

To ensure the reliability and specificity of results when working with Phospho-PRKDC (Thr2609) antibody, the following controls should be included:

Positive Controls:

• Cells treated with ionizing radiation (2-10 Gy, analyzed 30-60 minutes post-treatment) should show increased Thr2609 phosphorylation

• Cell lines known to express DNA-PKcs (e.g., HeLa, Fus1) treated with DNA-damaging agents like etoposide (VP-16)

Negative Controls:

• Untreated cells should show minimal phosphorylation

• DNA-PKcs deficient cell lines (e.g., M059J) should show no signal

• Cells treated with DNA-PK inhibitors (e.g., NU7441) prior to damage induction

• Phosphatase treatment of lysates should eliminate the signal

Specificity Controls:

• Peptide competition assay using phosphorylated vs. non-phosphorylated peptides

• Cells expressing T2609A mutant DNA-PKcs should not be recognized by the antibody after DNA damage

Loading Controls:

• Total DNA-PKcs antibody on parallel samples to normalize for total protein expression

• Standard loading controls (e.g., actin, GAPDH) for whole cell lysates

• Nuclear protein controls (e.g., lamin, histone H3) for nuclear extracts

Protocol Controls:

• Time course samples to capture the optimal window for phosphorylation detection (10 min to 4 hours post-damage)

• Dose-response samples to determine sensitivity (2-10 Gy is typically sufficient)

How can Phospho-PRKDC (Thr2609) antibody distinguish between DNA damage-induced vs. cytokine-induced phosphorylation?

Recent research has revealed that DNA-PKcs can be phosphorylated at Thr2609 in response to inflammatory cytokines like TNF-α, independent of DNA damage . Distinguishing between these two activation mechanisms requires strategic experimental approaches:

Differential Kinetics Analysis:

• DNA damage-induced phosphorylation typically peaks around 30-60 minutes after damage and persists for 4-6 hours

• Cytokine-induced phosphorylation can occur more rapidly (within 5 minutes of TNF-α treatment) and may have different persistence patterns

Co-treatment Experiments:

• Pre-treat cells with DNA-PK inhibitors (e.g., NU7441) to block autophosphorylation

• Use p38MAPK inhibitors, which can block TNF-α-induced but not DNA damage-induced PRKDC phosphorylation

Mechanistic Separation:

• In Ku70/80-deficient cells, DNA damage-induced phosphorylation of Thr2609 is impaired, while TNF-α-induced phosphorylation remains intact

• Use immunoprecipitation to examine co-association with different protein complexes (DNA damage response proteins vs. inflammatory signaling components)

Co-localization Studies:

• DNA damage-induced phospho-PRKDC typically forms discrete nuclear foci that co-localize with other DNA damage markers (e.g., γH2AX)

• Cytokine-induced phosphorylation may show different nuclear distribution patterns

What are the kinetics of Thr2609 phosphorylation after DNA damage and how should experiments be designed to capture this?

The phosphorylation of DNA-PKcs at Thr2609 follows specific temporal dynamics after DNA damage that are important to consider in experimental design:

Temporal Profile:

• Detectable as early as 10 minutes post-irradiation

• Peaks at approximately 30-60 minutes

• Persists for up to 4 hours

• Returns to basal levels by 6 hours

Experimental Design Considerations:

Time Point Selection:

• For maximum signal: collect samples at 30-60 minutes post-damage

• For early events: include 5, 10, and 15-minute time points

• For resolution of dynamics: collect samples at 0, 10, 30, 60, 120, 240, and 360 minutes

Damage Induction Methods:

• Ionizing radiation: provides uniform, simultaneous damage (2-10 Gy recommended)

• Radiomimetic drugs (e.g., etoposide/VP-16): longer-acting but may have different kinetics

• Laser microirradiation: allows real-time imaging of recruitment and phosphorylation

Detection Methods:

• Western blotting: quantitative assessment of total cellular phosphorylation

• Immunofluorescence: spatial resolution of phosphorylation events and foci formation

• Flow cytometry: single-cell analysis of phosphorylation across populations

How does Thr2609 phosphorylation relate to other phosphorylation sites on PRKDC?

DNA-PKcs undergoes phosphorylation at multiple sites, with complex interrelationships that affect its function in DNA repair:

Major Phosphorylation Clusters:

• ABCDE cluster: includes Thr2609, Thr2638, and Thr2647

• PQR cluster: includes Ser2056

Functional Differences:

• Thr2609 phosphorylation (ABCDE cluster) promotes access to DNA ends and affects repair pathway choice

• Ser2056 phosphorylation (PQR cluster) facilitates release of DNA-PKcs from DNA ends

Temporal Patterns:

• Both sites are rapidly phosphorylated after DNA damage

• They may have different dephosphorylation kinetics

Pathway Dependencies:

• Thr2609 can be phosphorylated both by DNA-PKcs itself (autophosphorylation) and by ATM in some contexts

• Ser2056 phosphorylation is primarily DNA-PKcs-dependent autophosphorylation

Inflammatory Response:

• Both Ser2056 and Ser2612 (equivalent to Thr2609 in human) can be phosphorylated in response to TNF-α

• The pattern of phosphorylation may differ between DNA damage and inflammatory stimuli

Biological Significance:

• Mutations at Thr2609 cause stronger phenotypes in some contexts than mutations at Ser2056

• The ABCDE cluster (including Thr2609) is important for hematopoietic development and affects repair pathway choice (cNHEJ vs. Alt-EJ)

• The T2609 cluster phosphorylation plays a role in promoting classical NHEJ repair pathway choice during class switch recombination

What methods can be used to study the functional consequences of Thr2609 phosphorylation?

Understanding the functional impact of Thr2609 phosphorylation requires multiple complementary approaches:

Genetic Models:

• T2609A knock-in mouse models: Allows evaluation of in vivo consequences in a physiological context

• CRISPR/Cas9-mediated T2609A mutation in cell lines: For cellular studies with endogenous expression levels

Cellular Assays:

• Clonogenic survival assays: Measures cell viability after DNA damage

• DSB repair assays: Such as pulsed-field gel electrophoresis (PFGE) or Fraction of Activity Released (FAR) assay to assess repair kinetics

• Immunofluorescence for repair factors: To study recruitment/retention of other repair proteins

• Class switch recombination assays: In B cells to assess impact on physiological DSB repair

Molecular Techniques:

• Chromatin immunoprecipitation (ChIP): To analyze recruitment of DNA-PKcs and other factors to break sites

• High-throughput sequencing of repair junctions: To characterize repair pathway choice (e.g., HTGTS method used to analyze CSR junctions)

• In vitro kinase assays: To assess how phosphorylation affects DNA-PKcs kinase activity

Pharmacological Interventions:

• DNA-PK inhibitors: To distinguish kinase-dependent vs. phosphorylation-dependent effects

• Phosphatase inhibitors: To maintain phosphorylation status for extended analysis

What cell type-specific variations should be considered when using Phospho-PRKDC (Thr2609) antibody?

Different cell types may exhibit variations in DNA-PKcs phosphorylation patterns and responses that researchers should consider:

Cell Type-Specific Expression Levels:

Functional Variations:

• B cells rely on DNA-PKcs for class switch recombination, where T2609 phosphorylation affects repair pathway choice (cNHEJ vs. Alt-EJ)

• Endothelial cells show robust TNF-α-induced phosphorylation of Thr2609 independent of DNA damage

• Cancer cells may show constitutive phosphorylation due to genomic instability or altered signaling pathways

Technical Considerations:

• Nuclear extraction efficiency varies between cell types

• Fixation protocols may need optimization for specific cell types

• Background levels and non-specific binding patterns can differ between tissues

Experimental Design Adjustments:

• Cell type-specific antibody dilutions may be required

• Damage induction methods may need adjustment (radiation dose, drug concentration)

• Different time points for peak phosphorylation detection might be necessary

How can Phospho-PRKDC (Thr2609) antibody be validated for specificity?

Thorough validation of Phospho-PRKDC (Thr2609) antibody specificity is critical for reliable results:

Peptide Competition Assay:

• Pre-incubate antibody with:

  • Phosphorylated peptide (should block signal)

  • Non-phosphorylated peptide (should not block signal)

  • Unrelated phospho-peptide (should not block signal)

• Use these pre-absorbed antibodies in Western blot or immunostaining

• Signal should be abolished only with the specific phospho-peptide

Genetic Validation:

• Compare signal between:

  • Wild-type cells

  • DNA-PKcs knockout cells (e.g., M059J)

  • Cells expressing T2609A mutant (should show no phospho-signal)

• Complement deficient cells with wild-type DNA-PKcs (should restore signal)

Pharmacological Validation:

• Treat cells with:

  • DNA-PK inhibitors before damage (should reduce signal)

  • Lambda phosphatase (should eliminate signal)

  • ATM/ATR inhibitors (may partially reduce signal in some contexts)

• Compare signal reduction patterns

Damage-Inducible Response:

• Establish a dose-response relationship (2-10 Gy)

• Demonstrate time-dependent induction and resolution

• Compare multiple DNA damage inducers (IR, VP-16, H₂O₂)

Multiple Antibody Comparison:

• Test multiple antibodies against the same epitope from different vendors

• Compare signal patterns and specificity profiles

• Use monoclonal and polyclonal antibodies in parallel

What are the optimal sample preparation methods for detecting Phospho-PRKDC (Thr2609)?

Sample preparation is critical for preserving phosphorylation status and obtaining reliable results:

For Western Blot Analysis:

Cell Lysis Protocol:

• Place cells on ice immediately after treatment

• Wash once with ice-cold PBS containing phosphatase inhibitors

• Lyse cells in buffer containing:

  • 50 mM Tris-HCl (pH 7.4)

  • 150 mM NaCl

  • 1% NP-40 or Triton X-100

  • 0.5% sodium deoxycholate

  • Protease inhibitor cocktail

  • Phosphatase inhibitor cocktail (critical)

  • 1 mM DTT

• Incubate on ice for 30 minutes with occasional vortexing

• Centrifuge at 14,000 × g for 15 minutes at 4°C

• Collect supernatant and determine protein concentration

Nuclear Extract Preparation (Preferred Method):

• Wash cells with ice-cold PBS containing phosphatase inhibitors

• Resuspend in hypotonic buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA)

• Add NP-40 to 0.5% final concentration

• Centrifuge to collect nuclei

• Extract nuclear proteins with high-salt buffer (20 mM HEPES pH 7.9, 0.4 M NaCl, 1 mM EDTA)

• Include phosphatase inhibitors throughout

For Immunofluorescence:

Fixation Protocol:

• Wash cells with PBS

• Fix with 4% paraformaldehyde for 10 minutes at room temperature

• Alternatively, use methanol fixation (-20°C for 10 minutes) which may better preserve some phospho-epitopes

• Permeabilize with 0.2% Triton X-100 in PBS for 5 minutes

• Block with 5% BSA in PBS containing phosphatase inhibitors

For Tissue Samples:

FFPE Tissue Processing:

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

• Process and embed in paraffin following standard protocols

• Cut 4-5 μm sections

• Perform antigen retrieval:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0)

  • Pressure cooker method preferred (120°C for 10 minutes)

• Block endogenous peroxidase activity if using HRP detection

What are the potential research applications of Phospho-PRKDC (Thr2609) antibody beyond DNA damage studies?

Phospho-PRKDC (Thr2609) antibody has applications beyond traditional DNA damage research:

Inflammatory Response Research:

• Monitoring DNA-PKcs activation in response to inflammatory cytokines such as TNF-α

• Studying cross-talk between inflammatory signaling and DNA repair mechanisms

• Investigating the role of DNA-PKcs in inflammatory diseases

Cancer Research:

• Evaluating DNA-PKcs activation status in tumor samples

• Assessing response to radiation therapy or chemotherapeutics

• Developing biomarkers for treatment resistance or sensitivity

• Monitoring effects of DNA-PK inhibitors in clinical trials

Neurodegenerative Disease Studies:

• Investigating DNA-PKcs activation in models of neurodegeneration

• Studying the role of DNA damage and repair in neurodegenerative conditions

• Monitoring neuronal stress responses

Developmental Biology:

• Analyzing DNA-PKcs activation during embryonic development

• Studying its role in hematopoietic development and immune system formation

• Investigating cell type-specific DNA repair mechanisms during differentiation

Aging Research:

• Monitoring changes in DNA-PKcs phosphorylation during aging

• Studying the relationship between DNA damage accumulation and senescence

• Investigating interventions that affect DNA repair efficiency

Drug Discovery:

• Screening compounds that modulate DNA-PKcs phosphorylation

• Developing assays for DNA-PK inhibitor efficacy

• Investigating off-target effects of kinase inhibitors

What are common technical issues when using Phospho-PRKDC (Thr2609) antibody and how can they be resolved?

How can experimental artifacts be distinguished from genuine Thr2609 phosphorylation?

Distinguishing true phosphorylation signals from artifacts requires systematic controls:

Biological Controls:

• DNA-PKcs knockout or knockdown cells should show no specific signal

• T2609A mutant-expressing cells should show no phospho-specific signal

• Dose-dependent increase with DNA damage inducers (2-10 Gy)

• Time-dependent phosphorylation and dephosphorylation kinetics (peaks at 30-60 min)

Technical Controls:

• Lambda phosphatase treatment of lysates should eliminate phospho-specific signal

• Peptide competition with phospho-peptide should abolish signal

• Phospho-independent DNA-PKcs antibody should detect total protein regardless of phosphorylation status

• Multiple phospho-specific antibodies targeting different epitopes should show consistent patterns

Experimental Design Controls:

• Include unstressed cells to establish baseline

• Perform parallel detection of other DNA damage markers (γH2AX)

• Compare results from multiple detection methods (WB, IF, IP)

• Include cell cycle analysis to account for cell cycle-dependent variations