Figure 5: RNF20–RNF40 regulates translocation of TRAIP to DSBs.

(a) Schematic structure of TRAIP and RNF20. The grey line below TRAIP indicates bait, and the grey line above TRAIP highlights the prey clone identified in the yeast two-hybrid screen. (b) Cell extracts from 293 T cells expressing Myc-TRAIP and SFB-RNF20 were immunoprecipitated with anti-Flag antibody and then analysed using immunoblotting. (c) The interaction between endogenous TRAIP and RNF20. 293 T cells were exposed to 0 or 10 Gy of ionizing radiation and harvested after 1 h. Immunoprecipitation was performed using control IgG or anti-RNF20 antibodies and then analysed using immunoblotting. (d) Cell extracts from 293 T cells expressing indicated proteins were immunoprecipitated with anti-Flag antibody and then analysed by immunoblotting. (e,f) Domains of TRAIP required for TRAIP–RNF20 or TRAIP-RNF40 interactions. 293 T cells were transfected with plasmids encoding GFP-RNF20 or GFP-RNF40 together with WT TRAIP or serial deletion mutants. Cell lysates were subjected to immunoprecipitation with anti-FLAG antibody and then analysed by immunoblotting. (g) 293 T cells were transfected with plasmids encoding SFB-RNF20 with Myc-TRAIP WT or D6 mutant. After 24 h, cell lysates were subjected to pulldown with streptavidin beads and immunoblotted with the indicated antibodies. (h) U2OS cells expressing GFP–TRAIP were transfected with siRNF20 or siRNF20 and siRNA-resistant RNF20 expression vector. 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 graphed in the bottom panel. Experimental strategy is illustrated. Error bars indicate the s.d. (i) The 293 T cells were transfected with control, RNF20 or RNF40 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. (j) Schematic illustration of the binding domain architecture of RNF20/40, TRAIP and RAP80. See full blots in the Supplementary Fig. 14.