Fig. 2.

Smad7 enhances the activity of ATM. a The expression of phospho-ATM was examined in control and S7OE A549 cells at the indicated time points after treatment with 200 ng/ml NCS. The anti-Flag antibody was used to detect Flag-tagged Smad7. Equal protein loading was confirmed by α-tubulin detection. Ratio pATM/ATM was calculated by densitometric analysis. b Control or S7OE A549 cells were treated with 200 ng/ml NCS, and the expression of γH2AX was examined at the indicated time points. Smad7 antibody was used to detect Smad7 overexpression. α-tubulin was used as a loading control. c Phospho-ATM nuclear foci formation was assessed in control and S7OE A549 and NCI-H292 cells at the indicated time points after treatment with 100 ng/ml NCS. DAPI was used for nuclear staining. d The ATM phosphorylation in Smad7^+/− and Smad7^−/− MEF cells was examined at the indicated time points after treatment with 200 ng/ml NCS. Endogenous Smad7 levels were assessed using an anti-Smad7 antibody. Equal protein loading was confirmed by α-tubulin detection. e The activation of H2AX was examined in Smad7^+/− and Smad7^−/− MEF cells at the indicated time points upon 200 ng/ml NCS treatment. Endogenous Smad7 levels were detected using an anti-Smad7 antibody. Equal protein loading was confirmed by α-tubulin detection. f Smad7^+/− and Smad7^−/− MEF cells were treated with 100 ng/ml NCS for 30 min. The cells were then fixed and incubated with a phospho-ATM-specific antibody to stain the nuclear foci and DAPI to stain the nuclear DNA