FIGURE 3.

PRT4165 does not inhibit the E3 ubiquitin ligase activity of RNF8 in vitro. A, effect of PRT4165 on the E3 ubiquitin ligase activity of RNF8/RNF168 in vitro. Ubiquitin E3 ligase activity was investigated by incubating histone H2A substrate with RNF8/RNF168 in the presence of E1 and E2 enzymes. Reactions were carried out in the presence of different concentrations of the PRT4165 inhibitor with a 30-min preincubation step as described under “Experimental Procedures.” H2A ubiquitylation products were analyzed by SDS-PAGE and visualized using an H2A antibody. Quantifications of the immunoblots from two independent experiments were done using Odyssey software and were plotted on the right using Prism Software. B, effect of PRT4165 on MDC1 IRIF. U2OS cells were either mock treated or treated with 50 μm of the PRT4165 for 5 min before they were exposed to radiation (2 Gy). The cells were allowed to recover for 30 min in the presence of the PRT4165. The cells were then fixed and stained as indicated. The average number of MDC1 IRIF per cell was quantified and plotted as indicated. C, effect of PRT4165 on RNF8/RNF168 IRIF. U2OS cells transiently transfected with either GFP-RNF8 or GFP-RNF168. Medium and low GFP-expressing cells were selected by G418. The cells were either mock treated or treated with 50 μm of the PRT4165 for 5 min before they were exposed to radiation (2 Gy). The cells were allowed to recover for 30 min in the presence of the PRT4165. The cells were then fixed and stained as indicated. The GFP signal was used to monitor the RNF8/RNF168 IRIF. The average number of GFP-RNF8/GFP-RNF168 IRIF per cell was quantified and plotted as indicated. The scale bars represent 10 μm.