Journal:

Article Title: DNA-PKcs function regulated specifically by protein phosphatase 5

doi: 10.1073/pnas.0307765100

Figure Lengend Snippet: Phosphorylation status of DNA–PKcs after irradiation in PP5 overexpressing clones. PP5.C4 and PP5.C13 subclones were irradiated with IR (11.5 Gy) and recovered for 30–410 min. The undamaged control (UD) is shown on the left. Nuclear extracts were prepared, and 20–60 μg of protein was applied to a SDS gel. After Western blotting, the membrane was stained with rabbit antibody to DNA–PKcs pT2609 (A), rabbit antibody to DNA–PKcs pS2056 (B), or rabbit antibody to ATM pS1981 (C). For loading controls the same membranes were stripped and incubated with antibody to total DNA–PKcs or to DNA polymerase δ. PP5 overexpresssion was visualized by anti-PP5 antibody. * and ** denote phosphorylated DNA–PKcs degradation products.

Article Snippet: The proteins were transferred by Western blotting onto a nitrocellulose membrane (Bio-Rad) and incubated with antibody 18-2 to DNA–PKcs (Neomarkers, Fremont, CA), antibody to pT2609, antibody to pS2056 site, antibody to DNA polymerase δ (Transduction Laboratories, Lexington, KY), antibody to PP5 (Transduction Laboratories), or antibody to ATM pS1981 (Rockland, Gilbertsville, PA).

Techniques: Phospho-proteomics, Irradiation, Clone Assay, Control, SDS-Gel, Western Blot, Membrane, Staining, Incubation