The CSK+R extraction protocol reveals Ku at DNA ends generated by various DNA-damaging agents. (A) U2OS cells were untreated (left columns) or irradiated with 10 Gy of IR (right columns), postincubated for 5 min, and fixed without CSK or with CSK or CSK+R preextraction. Quantifications of Ku foci generated 5 min after irradiation are provided in Fig. 5 A. (B) U2OS cells stably expressing GFP-FLAG-XRCC4 (clone 01) were untreated (NT) or treated with 10 Gy of IR, postincubated for 5 min, preextracted with CSK+R, and processed for immunofluorescence with an anti-GFP antibody to boost the GFP signal. (C) U2OS cells were untreated (top row, DMSO) or treated for 30 min with 100 µM etoposide (middle and bottom rows) before being preextracted with CSK+R and processed for immunofluorescence. (D) U2OS Tet-On cells were transiently transfected with an empty plasmid (pICE; control) or with a plasmid expressing HA-I-PpoI (pICE-HA-NLS-I-PpoI; I-PpoI), and after 24 h, I-PpoI expression was induced by doxycycline for 5 h. Whole-cell extracts were collected and analyzed by immunoblotting (top), and cells on coverslips were preextracted with CSK+R and analyzed by immunofluorescence (bottom). In this figure, insets represent a twofold zoom to highlight Ku80 microfoci (boxed regions). The position of each nucleus, as defined by DAPI staining, is highlighted by a dotted line. Bars: (white) 10 µm; (green) 1 µm.