Figure 3: TRAIP is critical for RAP80 recruitment to the sites of DNA lesions.

(a–c) U2OS cells expressing GFP–RAP80 or GFP–TRAIP were transfected with indicated siRNAs or siRNAs together with siRNA-resistant TRAIP WT, TRAIP-D1, -D2 or -D6. After 72 h, cells were subjected to laser microirradiation. Laser stripes were examined at the indicated time points. The intensity of each laser stripe in each time point was determined by averaging values from 10 cells and was graphed in the right panel. Experimental strategy is illustrated. (d) Cell fractionation to determine protein localization in response to DNA damage. The 293 T cells were transfected with control, RAP80 or TRAIP siRNA. After 48 h, the transfected cells were exposed to 0 or 10 Gy of ionizing radiation for 1 h. The chromatin fractions were subjected to western blot analysis. (e) Western blot analysis for TRAIP or RAP80 protein level in 293 T cells transfected with indicated siRNAs. (f) Kinetics of GFP–TRAIP or GFP–RAP80 translocation to DSBs. Average intensity of the laser stripes from 10 cells was presented with graph. (g,h) GFP-tagged RAP80 WT or indicated mutant was individually transfected into U2OS cells. After 72 h, the cells were subjected to laser microirradiation. The intensity of each laser stripe in each time point was determined by averaging values from 10 cells. Error bars indicate the s.d. See full blots in the Supplementary Fig. 14.