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. Author manuscript; available in PMC: 2011 Mar 28.
Published in final edited form as: Nat Chem Biol. 2008 Jan 6;4(2):119–125. doi: 10.1038/nchembio.63

Figure 2.

Figure 2

Mirin inhibits MRN-dependent activation of ATM. (a) Mirin inhibits ATM-dependent phosphorylation of Nbs1 and Chk2. X. laevis extracts were incubated with DMSO or mirin as indicated and then treated with DSBs. Nbs1 (upper panel) and Chk2 (lower panel) electrophoretic mobility were monitored by western blot. (b) Mirin inhibits MRN-dependent activation of ATM. Mock-treated (dark gray) or Mre11-depleted (light gray) extracts were incubated with 100 μM of mirin (lanes 3, 5, 7 and 9). ATM activation was triggered by addition of DSB-containing DNA at the indicated concentrations. Following DNA pull-down, ATM activation was assayed in the soluble fractions by H2AX peptide phosphorylation, or by western blot for phospho-Ser1981 of ATM (P-ATM). Each bar represents the average of four different experiments with s.d. shown. Values marked with asterisks are significantly different (P < 0.003). Mre11 depletion is shown below the graph. (c) Mirin prevents MRN- and DNA-dependent activation of dimeric ATM in vitro. Dimeric ATM activity was assayed in the presence of DNA (lane 2), MRN (lane 3), MRN and DNA (lanes 4 to 7), and with or without mirin (lanes 5 and 6). ATM activation was monitored by western analysis of Ser15-phosphorylated p53. (d) Mirin does not inhibit the activity of monomeric ATM in vitro. Monomeric ATM activity was assayed as in c in the presence of mirin, as indicated. WT, wild type.