Figure 3. Concerted activation of DNA damage signaling by DNA-PKcs, ATM, and ATR. (A) DNA-PKcs, ATM, and ATR bind to the biotinylated DNA substrates. DNA structures biotinylated at one DNA end were coupled to streptavidin-coated beads and then incubated with nuclear extracts for 10 min at 20°C, in the absence or presence of the DNA-PKcs inhibitor IC86621 (100 µM), the ATM inhibitor KU-55933 (10 µM), or the ATR inhibitor ETP-46464 (1 µM), as indicated. DNA-bound DNA-PKcs, ATM, and ATR were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (B) DNA-bound TopBP1 and Chk1 were isolated and detected as described in A. (C) Nuclear extracts prepared from cells treated with control shRNA or ATR shRNA were incubated with the indicated biotinylated DNA substrates, as described in A. ATR, DNA-PKcs, and Chk1 proteins were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (D) Nuclear extracts from cells treated with control shRNA or RPA shRNA were incubated for the indicated time periods with gDNA and probed for the indicated proteins by Western blotting. iDNA-PKcs, inhibitor of DNA-PKcs; iATM, inhibitor of ATM; iATR, inhibitor of ATR.
Figure 3.

Concerted activation of DNA damage signaling by DNA-PKcs, ATM, and ATR. (A) DNA-PKcs, ATM, and ATR bind to the biotinylated DNA substrates. DNA structures biotinylated at one DNA end were coupled to streptavidin-coated beads and then incubated with nuclear extracts for 10 min at 20°C, in the absence or presence of the DNA-PKcs inhibitor IC86621 (100 µM), the ATM inhibitor KU-55933 (10 µM), or the ATR inhibitor ETP-46464 (1 µM), as indicated. DNA-bound DNA-PKcs, ATM, and ATR were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (B) DNA-bound TopBP1 and Chk1 were isolated and detected as described in A. (C) Nuclear extracts prepared from cells treated with control shRNA or ATR shRNA were incubated with the indicated biotinylated DNA substrates, as described in A. ATR, DNA-PKcs, and Chk1 proteins were pulled down with streptavidin-coated beads, resolved by SDS-PAGE, and detected by Western blotting. (D) Nuclear extracts from cells treated with control shRNA or RPA shRNA were incubated for the indicated time periods with gDNA and probed for the indicated proteins by Western blotting. iDNA-PKcs, inhibitor of DNA-PKcs; iATM, inhibitor of ATM; iATR, inhibitor of ATR.

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