Differential Dynamics of ATR-Mediated Checkpoint Regulators
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 UTR of Rad9 for the indicated time periods. Cell extracts were prepared and western blot analysis was performed using the indicated antibodies.
