DNA-PKcs is essential for DNA damage signal activation. (A) Nuclear extracts prepared from cells treated with control shRNA (shControl) or DNA-PKcs shRNA were incubated without DNA, with dsDNA, or with gDNA, and the indicated proteins were detected by Western blotting. (B) Nuclear extracts from HCT116 and HCT116 DNA-PKcs^−/− cells were incubated with gDNA, and HCT116 DNA-PKcs^−/− nuclear extracts were complemented with increasing amounts of DNA-PK purified from HeLa cells (0.025, 0.05, 0.1, 0.2, and 1 U/µl). (C) Nuclear extracts from cells treated with control shRNA or Ku70 shRNA were incubated for the indicated time periods with gDNA and probed for the indicated proteins by Western blotting. (D) Biotinylated DNA substrates were incubated with nuclear extracts in the absence or presence of IC86621 and pulled down, and the indicated DNA-bound proteins were analyzed by Western blotting. (E) Biotinylated DNA substrates were incubated with nuclear extracts from cells treated with control shRNA or ATR shRNA, isolated, and probed for the indicated proteins.