Figure 4

Rac1 inhibition abolishes IR-induced activation of both ATM and ATR signaling. (A) MCF-7 cells were treated with/without 20-Gy IR in the presence or absence of 100 μM NSC23766 and incubated for 1 hour at 37°C before analysis. To assess the ATM kinase activity, ATM was immunoprecipitated from cell lysate by using anti-ATM antibody (2C1) and assayed for ATM activity by using p53 recombinant protein as substrate. To measure the Chk2 activity, Chk2 was immunoprecipitated from cell lysate by using B-4 anti-Chk2 antibody and assayed for Chk2 activity by using Cdc25C recombinant protein as substrate. As controls, ATM and Chk2 protein levels in the immunoprecipitates (IP-WB) as well as in cell lysates (WB) were assessed with immunoblotting. (B) ATR and Chk1 were immunoprecipitated from cell lysates by using N-19 anti-ATR and G-4 anti-Chk1 antibody, respectively. ATR activity was assayed by using p53 recombinant protein substrate, and Chk1 activity assayed by using Cdc25C recombinant protein substrate. As controls, ATR and Chk1 protein levels in the immunoprecipitates (IP-WB), as well as in cell lysates (WB) were assessed with immunoblotting. (C) MCF-7 cells were exposed to IR at the indicated doses in the presence or absence of NSC23766, incubated for 1 hour, and assessed for Chk1 and Chk2 activities. *Kinase assay does not contain Cdc25C substrate. (D) T47D and ZR-75-1 cells were exposed to 10-Gy IR in the presence or absence of 100 μM NSC23766, incubated for 1 hour, and analyzed for Chk1 and Chk2 activities.