Figure 2: Subcellular translocalization of TRAIP to the DNA-damage site.

(a) U2OS cells expressing GFP–TRAIP were subjected to laser microirradiation. The laser stripes were examined for 10 min after the microirradiation. The percentage of cells with positive GFP on the laser stripes was determined. More than 50 nuclei per condition. (b) U2OS cells were treated with laser microirradiation. After 30 min, the cells were fixed and stained with anti-TRAIP and -γH2AX antibodies. (c) U2OS cells expressing indicated proteins were subjected to laser microirradiation (top), and the cells with positive GFP on laser stripes (10 min after laser microirradiation) were presented with bar graph. More than 50 cells in each experiment. (d) mCherry-LacI-FokI was co-transfected with indicated GFP-tagged expression vector into U2OS-DSB reporter cells. After 48 h, live cell imaging was performed with confocal microscopy. GFP-positive cells over total counts were denoted. (e) U2OS cells expressing GFP–TRAIP and RFP-RAP80 were subjected to laser microirradiation. Colocalization of TRAIP with RAP80 at laser-induced DNA lesions (10 min after laser microirradiation). (f) Immunofluorescence assays were performed with 293 T cells expressing Flag-RAP80 and Myc-TRAIP. Cells with TRAIP and RAP80 positive were counted and presented with the bar graph. More than 50 nuclei per condition. (g) Overexpressed TRAIP recruits the endogenous RAP80 to the nuclear sparkle. Immunofluorescence assays were performed with 293 T cells expressing Myc-TRAIP using anti-Myc and anti-RAP80 antibodies. Percentage of RAP80-positive cells was determined. More than 50 nuclei per condition. DAPI was used as an indicator for the nucleus. The results represent the average of three independent experiments in each comparison. Error bars indicate the s.d.