Fig. 2. Rad17 is required for Rad9 to form foci in response to DNA damage. (A) HeLa cells were transfected with siRNA oligonucleotides as indicated and were left untreated or exposed to aphidicolin. Whole cell extracts were prepared for western blotting (left panel) and the percentage of cells exhibiting more than ten Rad9 foci was determined (right panel). (B) U2OS cells expressing GFP-Rad9 were transfected with empty vector, or vector containing Flag-Rad17 or Flag-Rad17^AA. Cells were left untreated or exposed to aphidicolin. Chromatin fractions were prepared and extracts subjected to western blotting using antibodies as indicated. (C) U2OS cells stably expressing GFP-Rad9 were transfected with Flag-Rad17 or Flag-Rad17^AA constructs prior to treating cells with aphidicolin. Flag-positive (transfected) or Flag-negative (untransfected) cells were identified by immunofluorescence and cells scored for GFP-Rad9 foci. (D) U2OS cells stably expressing GFP-Rad9 were transfected as in C, after which cells were treated with UV. Flag-positive (transfected) or Flag-negative (untransfected) cells were identified by immunofluorescence and cells were scored for GFP-Rad9 foci.