Figure 2

ATM is modified by poly(ADP)ribose. A: ATM was immunoprecipitated in G361 cells and the presence of poly(ADP ribose) was tested by immunoblot analysis in a time course, reaching a maximum after 15 min of IR (10 Gy). Equal loading was normalized by the input of ATM. B: Double indirect immunofluorescence in 3T3 fibroblasts (parp-1+/+) of poly(ADP-ribose) (red signal) and ATM (green). Yellow signal correspond with poly(ADP-ribosyl)ation of ATM. C: PARP-1 is needed for optimal activation of ATM. In vitro ATM-kinase assay. ATM activity increases after γ-irradiation in wild-type mouse splenocytes, but not in parp-1 knockout mouse splenocytes, where ATM is activated in control (upper panels). In G361 cells and C3ABR (middle and lower panel) irradiated with or without ANI co-treatment, ATM kinase activity increases after γ-irradiation and/or PARP inhibitors. Equal loading was checked with coomasie blue staining. Normalized signal respect to coomassie blue is shown below. Results are representative of three independent experiments.