Figure 2: Validation and functionality of stable cells expressing GFP-fusions. (a) Two independent U2OS clones expressing GFP-ATRIP were lysed after which immunoprecipitations were carried out using anti-GFP antibody. Western blot analysis of the immunoprecipitates using the indicated antibodies. (b) U2OS and Hela cells expressing GFP-Rad9 were seeded in low density and treated with different doses of UV. The number of surviving colonies after 10 days was counted. (c) Left panel: cells expressing GFP-ATRIP or control U2OS cells were left untreated or treated with UV. One hour later, cells were lysed and analyzed by western blotting with the indicated antibodies. Right panel: as left panel, but with two clones expressing GFP-Chk1. (d) Control U2OS cells or cells expressing GFP-Chk1 were left untreated or treated with 10 Gy IR. After 2 hours, cells were fixed and stained with PI and MPM2 and analyzed by FACS. The percentage of mitotic cells is indicated. (e) U2OS cells expressing GFP-Rad9 were transfected with siRNA oligos directed against Rad9 or the 3 UTR of Rad9 for the indicated time periods. Cell extracts were prepared and western blot analysis was performed using the indicated antibodies.