Phospho-NBN (Ser343) Antibody

What is the biological significance of NBN phosphorylation at Serine 343?

NBN (Nibrin) phosphorylation at Serine 343 represents a critical post-translational modification in the DNA damage response pathway. This phosphorylation is mediated by ATM kinase in response to ionizing radiation and is essential for proper intra-S phase checkpoint control and telomere maintenance . The phosphorylation at Ser343 enables NBN to participate effectively in the MRE11/RAD50/NBN (MRN) complex, which plays a crucial role in detecting and repairing DNA double-strand breaks. Studies have shown that this phosphorylation event is necessary for proper localization of the MRN complex to sites of DNA damage and for the activation of downstream signaling pathways that coordinate cell cycle arrest and DNA repair .

What is the difference between NBN, p95/NBS1, and Nibrin?

These are different names for the same protein:

• NBN is the official gene symbol

• p95 refers to the molecular weight of the protein (~95 kDa)

• NBS1 refers to the association with Nijmegen Breakage Syndrome

• Nibrin is another common name

The protein is a component of the MRE11/RAD50/NBN complex involved in DNA double-strand break repair and DNA damage-induced checkpoint activation . Mutations in the NBN gene are associated with Nijmegen breakage syndrome, characterized by microcephaly, growth retardation, immunodeficiency, and cancer predisposition .

How does phosphorylation of NBN at Ser343 change after DNA damage?

Upon DNA damage, particularly from ionizing radiation, ATM kinase is activated and rapidly phosphorylates NBN at Ser343. This can be observed as a slower electrophoretic mobility of NBN in gel electrophoresis . The MRC-5 cell line has demonstrated this mobility shift after IR exposure due to phosphorylation at both Ser278 and Ser343 residues .

The phosphorylation typically occurs within minutes of DNA damage and can persist for several hours, depending on the extent of damage and cell type. This modification is necessary for the proper assembly and localization of the MRN complex at DNA double-strand breaks and subsequent activation of downstream repair pathways .

What are the optimal cell treatment conditions to induce NBN Ser343 phosphorylation?

For robust induction of NBN Ser343 phosphorylation:

For experimental validation, include appropriate controls:

• Untreated cells (negative control)

• λ-phosphatase treatment (to confirm phospho-specificity)

• ATM inhibitors (e.g., KU-55933) to confirm kinase specificity

What experimental systems are most suitable for studying NBN Ser343 phosphorylation?

Based on published research, these experimental systems have proven effective:

• Cell lines: Human cell lines including MRC-5 fibroblasts, U2OS, HeLa, and lymphoblastoid cell lines show robust NBN phosphorylation responses . The PC-3M cell line has been validated for immunocytochemistry applications with Phospho-NBN (Ser343) antibodies .

• Primary cells: Primary human fibroblasts are suitable, particularly from patients with relevant DNA repair disorders for comparative studies.

• Tissue sections: Human testis tissue has shown clear immunohistochemical staining with Phospho-NBN (Ser343) antibodies, likely due to high expression levels of NBN in testicular tissue .

• In vitro systems: Bacterially expressed full-length NBN protein can be phosphorylated using mitotic cell extracts for biochemical studies .

How should NBN knockout or mutant models be designed for phospho-Ser343 studies?

When designing NBN knockout or mutant models:

• Complete knockout considerations:

  • Complete NBN knockout is embryonic lethal in mice

  • Use conditional knockout systems (e.g., Cre-loxP) for tissue-specific deletion

  • CRISPR/Cas9 with inducible systems for temporal control

• Phospho-site mutations:

  • S343A mutation: Prevents phosphorylation (phospho-dead)

  • S343D/E mutation: Phosphomimetic (simulates constitutive phosphorylation)

  • Consider generating both for comparative studies

• Disease-relevant mutations:

  • 657del5 mutation: Creates truncated forms (26 kDa and 70 kDa fragments)

  • Arg215Trp mutation: Affects the BRCT domain function

  • These mutations affect the MRN complex formation and DNA damage response differently

• Controls:

  • Include wild-type NBN re-expression in knockout backgrounds

  • Consider rescuing with phospho-mutants to demonstrate specificity

  • Use cells from Nijmegen breakage syndrome patients as disease models

What are the optimal protocols for Western blotting with Phospho-NBN (Ser343) antibodies?

Based on multiple validated protocols, here is an optimized Western blotting procedure:

Sample Preparation:

• Extract proteins using RIPA buffer supplemented with phosphatase inhibitors (10 mM NaF, 1 mM Na₃VO₄, 1 mM β-glycerophosphate)

• Include protease inhibitor cocktail

• Denature samples at 95°C for 5 minutes in Laemmli buffer

Gel Electrophoresis:

• Use 6-8% SDS-PAGE (NBN is ~95 kDa)

• Load 25-50 μg total protein per lane

• Include phosphatase-treated control

Transfer and Blocking:

• Transfer to PVDF membrane (recommended over nitrocellulose)

• Block in 5% BSA (not milk) in TBST for 1 hour at room temperature

Antibody Incubation:

• Primary antibody dilution: 1:500-1:1000 in 5% BSA/TBST

• Incubate overnight at 4°C

• Wash 3× in TBST, 5 minutes each

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

• Incubate 1 hour at room temperature

Detection:

• Use enhanced chemiluminescence (ECL) reagents

• Expected band: ~95 kDa

• Include total NBN antibody on stripped or parallel blot

Validation Controls:

• λ-phosphatase treatment of lysate

• ATM inhibitor-treated cells

• UV or IR-treated positive control

How should I validate the specificity of Phospho-NBN (Ser343) antibodies?

A comprehensive validation strategy should include:

• Phosphatase treatment:

  • Treat fixed cells or protein lysates with λ-phosphatase

  • This should abolish antibody recognition, confirming phospho-specificity

• Competitive peptide blocking:

  • Pre-incubate antibody with phospho-Ser343 peptide (S-L-S(p)-Q-G)

  • Should eliminate specific signal

  • Non-phosphorylated peptide should not block signal

• Mutant expression:

  • Express NBN S343A mutant (cannot be phosphorylated)

  • Compare with wild-type NBN

  • S343A should show no signal with the phospho-specific antibody

• ATM inhibition/knockout:

  • Treat cells with ATM inhibitors or use ATM-deficient cells

  • Should prevent Ser343 phosphorylation after DNA damage

• Immunodepletion:

  • Use chromatography with non-phosphopeptide to remove non-phospho specific antibodies

  • This approach has been used in commercial antibody production

What protocols yield optimal results for immunofluorescence with Phospho-NBN (Ser343) antibodies?

For optimal immunofluorescence staining:

Cell Preparation:

• Grow cells on glass coverslips

• Following treatment (e.g., IR), fix with 4% paraformaldehyde for 15 minutes

• Permeabilize with 0.2% Triton X-100 for 10 minutes

• Block with 5% BSA in PBS for 1 hour

Antibody Incubation:

• Primary antibody dilution: 1:50-1:200 in blocking buffer

• Incubate overnight at 4°C

• Wash 3× with PBS

• Secondary antibody: Anti-rabbit Alexa Fluor 488 (1:500)

• Include DAPI for nuclear counterstaining

Imaging Considerations:

• Phospho-NBN (Ser343) typically shows nuclear foci following DNA damage

• Co-stain with γ-H2AX to confirm localization at DNA damage sites

• For optimal visualization, use confocal microscopy

Controls:

• Untreated cells (minimal phosphorylation)

• λ-phosphatase-treated cells (should eliminate signal)

• Co-staining with total NBN antibody (different species) to confirm localization

This protocol has been validated in PC-3M cells, showing specific nuclear staining pattern following DNA damage .

How can I distinguish between specific and non-specific signals when using Phospho-NBN (Ser343) antibodies?

To differentiate specific from non-specific signals:

• True Phospho-NBN (Ser343) signals are characterized by:

  • A single band at approximately 95 kDa in Western blots

  • Inducibility by DNA damage (particularly IR)

  • Elimination by λ-phosphatase treatment

  • Absence in ATM-inhibited samples

  • Nuclear localization, often in distinct foci after DNA damage

• Common sources of non-specific signals:

  • Bands at unexpected molecular weights

  • Signals that persist after phosphatase treatment

  • Cytoplasmic staining (NBN is primarily nuclear)

  • Signal in NBN-knockout cells or Ser343Ala mutants

• Troubleshooting strategy:

  • If multiple bands appear, optimize blocking (use 5% BSA instead of milk)

  • Increase washing stringency (add 0.1% SDS to TBST)

  • Titrate antibody concentration

  • Perform peptide competition with phospho- and non-phospho-peptides

  • Include proper controls (ATM inhibitor, phosphatase treatment)

How should I interpret differences in NBN Ser343 phosphorylation patterns between different cell types?

When comparing phosphorylation patterns across cell types:

• Consider baseline ATM activity:

  • Some cancer cell lines have constitutively active ATM

  • Primary cells typically show lower basal phosphorylation

• Cell cycle distribution effects:

  • NBN phosphorylation varies through the cell cycle

  • Synchronize cells when comparing different cell types

  • S-phase cells may show higher baseline phosphorylation

• Expression level differences:

  • Normalize phospho-signal to total NBN levels

  • NBN is highly expressed in testis tissue

  • Expression can vary significantly between cell types

• Mutation status considerations:

  • Check for NBN mutations that might affect antibody binding

  • The 657del5 mutation creates truncated forms affecting phosphorylation

  • The Arg215Trp mutation affects BRCT domains and downstream signaling

• DNA damage response pathway integrity:

  • Defects in upstream kinases (ATM) will reduce phosphorylation

  • Alterations in other MRN complex components can affect NBN phosphorylation

  • Cancer cells often have altered DNA damage response pathways

How does the phosphorylation kinetics of NBN Ser343 correlate with other DNA damage response markers?

The temporal relationship between NBN Ser343 phosphorylation and other DDR markers:

For comprehensive analysis:

• Use time-course experiments after DNA damage

• Compare phosphorylation kinetics across multiple DDR proteins

• Consider the effects of different damage types (IR vs. replication stress)

• Analyze both the appearance and resolution of phosphorylation signals

How can Phospho-NBN (Ser343) antibodies be used to investigate cancer-specific alterations in the DNA damage response?

Phospho-NBN (Ser343) antibodies offer several approaches to study cancer-specific DDR alterations:

• Diagnostic applications:

  • Compare Ser343 phosphorylation levels in tumor vs. normal tissue

  • Correlate with tumor aggressiveness and treatment resistance

  • Use as a biomarker for ATM pathway functionality

• Therapeutic response monitoring:

  • Measure NBN Ser343 phosphorylation before and after radiotherapy

  • Assess DNA damage response activation following chemotherapy

  • Evaluate PARP inhibitor efficacy in combination therapies

• Resistance mechanism identification:

  • Analyze NBN phosphorylation in treatment-resistant vs. sensitive tumors

  • Investigate compensatory phosphorylation in ATM-deficient cancers

  • Study NBN phosphorylation in the context of homologous recombination defects

• Synthetic lethality approaches:

  • Screen for compounds that selectively affect cells with altered NBN phosphorylation

  • Identify vulnerabilities in tumors with MRN complex mutations

  • Develop combination strategies targeting parallel DNA repair pathways

Methodology should include:

• Tissue microarray analysis with phospho-specific antibodies

• Live-cell imaging with fluorescent reporters for dynamic studies

• Correlation with clinical outcomes and treatment responses

What are the most effective approaches for studying the dynamics of NBN Ser343 phosphorylation in living cells?

Advanced techniques for studying NBN Ser343 phosphorylation dynamics:

• FRET-based phosphorylation sensors:

  • Design NBN constructs with phospho-binding domains and fluorophore pairs

  • Monitor real-time phosphorylation in living cells

  • Enable single-cell analysis of phosphorylation kinetics

• NBN-GFP fusions with phospho-specific imaging:

  • Express NBN-GFP in cells

  • Fix at different timepoints after damage

  • Stain with phospho-specific antibodies

  • Quantify phosphorylation relative to total protein

• Laser microirradiation combined with live imaging:

  • Induce localized DNA damage with laser microirradiation

  • Track NBN recruitment and phosphorylation at damage sites

  • Correlate with other fluorescently-tagged DDR factors

• Complementary techniques:

  • FRAP (Fluorescence Recovery After Photobleaching) to study phosphorylation-dependent mobility

  • High-content screening to assess phosphorylation across many conditions

  • Single-molecule tracking to examine phosphorylation effects on NBN dynamics

Implementation considerations:

• Use phosphomimetic (S343D/E) and phospho-dead (S343A) mutants as controls

• Combine with ATM inhibitors to confirm kinase dependency

• Validate findings with fixed-cell immunofluorescence using phospho-specific antibodies

How can direct selection of monoclonal phosphospecific antibodies against NBN Ser343 improve experimental outcomes?

Novel approaches to phospho-specific antibody generation can overcome limitations:

• Advantages of direct selection methods:

  • No need for phosphoamino acid identification upfront

  • Use of full-length protein with natural phosphorylation

  • Selection of antibodies recognizing native conformation

  • Higher specificity for phosphorylated epitopes

• Implementation methodology:

  • Produce full-length NBN in bacteria

  • Phosphorylate using mitotic cell extracts containing natural kinases

  • Perform affinity-based antibody selection using scFv phagemid libraries

  • Select antibodies that specifically recognize the phosphorylated form

• Validation strategy:

  • Confirm phospho-specificity using λ-phosphatase treatment

  • Test against phospho-site mutants (S343A)

  • Verify recognition of endogenous protein by immunoblotting

  • Demonstrate loss of signal with phosphatase treatment

• Applications of improved antibodies:

  • More sensitive detection of low-abundance phosphorylated forms

  • Better performance in multiple applications (WB, IF, IHC, IP)

  • Reduced background and cross-reactivity

  • Improved reproducibility across experiments

This approach has been successfully demonstrated for other phosphoproteins, yielding highly functional monoclonal phosphospecific antibodies within one week .

What is the relationship between NBN Ser343 phosphorylation and the activation of the MRN complex in various genetic backgrounds?

The interplay between NBN phosphorylation and MRN complex function:

• Impact of NBS mutations on phosphorylation and function:

  • The 657del5 mutation produces a 70 kDa C-terminal fragment that can still be phosphorylated at Ser343

  • This fragment retains some capability to interact with MRE11 and γ-H2AX

  • The Arg215Trp mutation in the BRCT domain impairs MRN complex formation and shows reduced phosphorylation

  • These mutations affect the DNA damage response to different degrees

• Functional consequences across genetic backgrounds:

  • In wild-type cells: Phosphorylation of Ser343 is critical for proper MRN complex localization to DSBs

  • In NBS patient cells (657del5): The 70 kDa fragment shows some phosphorylation but impaired function

  • In ATM-deficient cells: Significantly reduced Ser343 phosphorylation correlates with defective checkpoint activation

  • In BRCA1/BRCA2-mutant cells: NBN phosphorylation occurs normally but downstream homologous recombination is impaired

• Methodological approach:

  • Compare NBN phosphorylation across cell lines with defined genetic backgrounds

  • Use complementation studies with wild-type and mutant NBN in knockout cells

  • Analyze MRN complex formation by co-immunoprecipitation

  • Measure functional outcomes (checkpoint activation, DNA repair efficiency)

  • Correlate phosphorylation levels with repair outcomes using Phospho-NBN (Ser343) antibodies