Phospho-DCLRE1C (S516) Antibody

What is DCLRE1C (Artemis) and why is phosphorylation at serine 516 significant?

DCLRE1C (DNA cross-link repair 1C), commonly known as Artemis, is a critical component of the non-homologous end-joining (NHEJ) pathway that repairs DNA double-strand breaks. It plays essential roles in:

• V(D)J recombination during lymphocyte development

• Hairpin opening during DNA repair processes

• Resolution of ionizing-radiation-induced DNA damage

Phosphorylation at serine 516 (S516) represents one of the four most rapid phosphorylation sites in Artemis (along with S534, S538, and S645) and is closely associated with its activation . This specific post-translational modification is critical for regulating Artemis function within the DNA repair machinery.

What applications are suitable for Phospho-DCLRE1C (S516) antibody detection methods?

Phospho-DCLRE1C (S516) antibody has been validated for multiple experimental applications:

These applications enable researchers to detect endogenous levels of DCLRE1C only when phosphorylated at S516, making it an excellent tool for studying activation states of this repair enzyme .

How should Phospho-DCLRE1C (S516) antibody be stored and handled for optimal performance?

For maximum stability and performance:

• Long-term storage: Store at -20°C for up to one year

• Short-term/frequent use: Store at 4°C for up to one month

• Avoid repeated freeze-thaw cycles

• Upon receipt, it is advisable to aliquot the antibody to minimize freeze-thaw cycles

• The antibody is typically supplied in PBS containing 50% glycerol, 0.5% BSA and 0.02% sodium azide at pH 7.4, which helps maintain stability

How can researchers validate the specificity of Phospho-DCLRE1C (S516) antibody in their experimental systems?

Validation of antibody specificity should include:

• Competitive inhibition assay: Use synthetic phosphopeptides matching the S516 region (e.g., VAGGS(p)QSPKLFS) to block antibody binding

• Mutation-based validation: Express Artemis mutation fragment (D37N-413) in cells (e.g., HeLa) as this acts as a competitive inhibitor of serine 516 phosphorylation

• Multiple sample testing: Validate across different cell lysates:

  • HEK293T cells

  • Mouse skeletal muscle tissues

  • Rat skeletal muscle tissues

• Phosphatase treatment control: Treat one sample set with lambda phosphatase to remove phosphorylation and confirm signal loss

What are optimal conditions for immunodetection of Phospho-DCLRE1C (S516) in different experimental contexts?

For Western Blotting:

• Recommended dilution: 1:500-1:1000

• Sample preparation: Standard SDS-PAGE gel electrophoresis

• Detection: Use appropriate secondary antibody (anti-rabbit IgG-HRP)

• Expected molecular weight: ~78 kDa

For Immunohistochemistry:

• Section thickness: 5-8 μm for OCT-embedded tissues

• Fixation: 4% paraformaldehyde (10 minutes)

• Blocking: Use 5% normal goat serum

• Primary antibody dilution: 1:50-1:200

• Incubation: Overnight at 4°C

• For human anagen hair follicle studies, a dilution of 1:500 has been empirically determined as optimal

For Immunofluorescence double-staining:

• Co-staining partners successfully used include:

  • Phospho-p53 (serine 15)

  • Bcl-2

  • Bax

  • Cytokeratins (10, 14, 16)

  • p21

  • c-myc

• DAPI counterstaining for nuclei visualization

How does Artemis S516 phosphorylation relate to other DNA damage response proteins?

Research has revealed complex interactions between phosphorylated Artemis and other DNA repair proteins:

• DNA-PKcs-mediated phosphorylation: DNA-PKcs phosphorylates Artemis at multiple sites including S516, along with S385, T410, S417, S503, S509, S518, S572, S589, T601, S645, T676, S679, S686, and T692

• ATM signaling pathway: While Artemis functions in NHEJ pathway, studies show that the Art-P70 and null alleles did not significantly impair ATM-dependent responses to DNA double-strand breaks, as measured by phosphorylation of ATM, H2AX, and KAP1

• MRN complex interactions: Research has examined how Artemis mutations affect the stability and localization of the MRN complex (Mre11, Rad50, and Nbs1), suggesting functional interplay between these repair factors

• V(D)J recombination: Artemis phosphorylation states influence its function during lymphocyte development, with experimental systems using Dclre1c−/− cells reconstituted with human DCLRE1C variants revealing how mutations affect recombination and DNA repair activities

What is the relationship between Artemis S516 phosphorylation and cellular processes such as apoptosis and proliferation?

Immunofluorescence studies in human hair follicles have revealed intriguing relationships between Artemis S516 phosphorylation and cellular fate determinants:

• Inverse relationship with phospho-p53:

  • Strong Artemis S516-P expression co-localized with weak phospho-p53 expression in the outer hair root sheath

  • This inverse pattern suggests Artemis S516-P may inhibit phospho-p53 expression, potentially reducing apoptosis while promoting cell survival

• Expression patterns in relation to apoptotic markers:

  • Artemis S516-P showed opposite expression trends to both pro-apoptotic Bax and anti-apoptotic Bcl-2

  • Artemis S516-P expression decreased gradually closer to the hair bulb, while Bax and Bcl-2 expression increased

• Co-expression with proliferation markers:

  • Artemis S516-P and c-myc showed similar expression patterns with both being reduced closer to the hair bulb

  • p21 was expressed close to the cortex and medulla where Artemis S516-P was positive

These findings suggest multifaceted roles for Artemis S516 phosphorylation in regulating cell survival, proliferation, and differentiation processes.

How can Phospho-DCLRE1C (S516) antibody be used to study SCID-related mutations?

Severe Combined Immunodeficiency (SCID) is frequently associated with DCLRE1C mutations. Researchers can employ Phospho-DCLRE1C (S516) antibody to:

• Characterize phosphorylation status in patient samples:

  • Compare phosphorylation levels between healthy controls and patients with DCLRE1C mutations

  • Correlate phosphorylation defects with clinical severity and immunological phenotype

• Functional complementation studies:

  • Use V-abl kinase transformed murine Dclre1c−/− A-MuLV pro-B cells engineered with a pMX-INV GFP recombination cassette

  • Transduce with retroviral vectors containing wild-type or mutant DCLRE1C cDNAs

  • Assess GFP expression as a readout of recombination activity

  • Examine γH2AX levels after irradiation to measure DNA repair capacity

• Mutation-specific effects on phosphorylation:

  • The table below shows examples of clinically relevant DCLRE1C mutations that could be studied for their impact on S516 phosphorylation:

What are the technical challenges in detecting low levels of phospho-Artemis (S516) in primary cells and tissues?

Researchers face several technical challenges when attempting to detect phospho-Artemis (S516) in primary cells:

• Transient nature of phosphorylation:

  • Phosphorylation events may be brief in response to DNA damage

  • Sample collection timing is critical for capturing the phosphorylation state

  • Consider using phosphatase inhibitors during sample preparation

• Cell type-specific expression:

  • Artemis expression varies significantly between tissue types

  • For human tissue studies, normal scalp specimens embedded in OCT and serial-sectioned at 5–8 μm thickness have been successful

  • Primary lymphocytes may require optimization of fixation protocols

• Antibody sensitivity optimization:

  • For immunohistochemistry in challenging samples, signal amplification methods may be necessary

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • For immunofluorescence, consider using tyramide signal amplification

• Control selection:

  • Include phosphatase-treated samples as negative controls

  • Use samples from DCLRE1C-deficient cells (if available) as specificity controls

  • Consider synthetic phosphopeptide blocking controls to validate signal specificity

How does understanding Artemis S516 phosphorylation contribute to our knowledge of DNA repair mechanisms and immune disorders?

The study of phospho-Artemis S516 has significant implications for multiple research areas:

• Mechanistic insights into DNA repair:

  • Phosphorylation at S516 represents a critical regulatory step in NHEJ pathway activation

  • Understanding this modification helps elucidate how cells respond to DNA damage

  • The temporal sequence of Artemis phosphorylation events provides insights into repair kinetics

• Implications for immunodeficiency disorders:

  • DCLRE1C mutations cause a spectrum of immunodeficiencies ranging from severe T−B−SCID to milder "leaky SCID"

  • Phosphorylation status may serve as a biomarker for disease severity or progression

  • Studies correlating phosphorylation with V(D)J recombination efficiency help explain clinical heterogeneity

• Potential therapeutic applications:

  • Targeting Artemis phosphorylation could modulate DNA repair capacity

  • This approach might sensitize cancer cells to radiation therapy

  • Alternatively, enhancing phosphorylation might benefit patients with hypomorphic DCLRE1C mutations

What emerging research questions remain regarding Artemis S516 phosphorylation?

Several important questions remain unanswered in this field:

• Regulatory mechanisms:

  • How is S516 phosphorylation coordinated with other post-translational modifications?

  • Which phosphatases regulate the dephosphorylation of this site?

  • Are there tissue-specific differences in phosphorylation regulation?

• Functional consequences:

  • Does S516 phosphorylation alter protein-protein interactions within repair complexes?

  • How does phosphorylation affect Artemis nuclease activity at a molecular level?

  • Are there differences in repair pathway choice based on phosphorylation status?

• Clinical correlations:

  • Can phospho-Artemis (S516) levels predict radiation sensitivity in cancer patients?

  • Do polymorphisms near S516 contribute to individual differences in DNA repair capacity?

  • Could phospho-Artemis (S516) serve as a diagnostic biomarker for subtle immunodeficiencies?