Fig. 3.

Smad7 interacts with ATM and Nbs1 upon DSB response and co-localizes with them at nuclear foci. a To determine whether Smad7 co-localized with Nbs1, Mre11, γH2AX or phospho-ATM at the site of DNA damage, S7OE A549 cells were treated with 50 ng/ml NCS for 1 h. b Whole cell lysates were prepared at the indicated time points after treatment with 200 ng/ml NCS in S7OE A549 and NCI-H292 cells. Flag-tagged Smad7 was pulled down using an anti-Flag antibody, and western blotting was performed using the indicated antibodies. c GST-tagged Smad7 was co-expressed with a Myc-tagged ATM fragment in 293T cells. ATM fragments described in supplemental material Fig. S4 were immunoprecipitated with the Myc antibody, and the interacting Smad7 was detected using an anti-GST antibody. d To confirm the Nbs1-Smad7 interaction, Smad7 was immunoprecipitated with the Flag-specific antibody and the HA-specific antibody was used to detect the interacting Nbs1. Cells were treated with 200 ng/ml NCS to induce DSB stress