Figure 6.

BRCA1 preferentially interacts with the N-terminal region of DNA-PKcs near the 2056 cluster. (A) FLAG-tagged N-terminal fragment (N-PKcs) and C-terminal fragment (C-PKcs) of DNA-PKcs were expressed and IPed from Sf9 cells using FLAG antibody, washed three times and the IP DNA-PKcs fragments still bound to the protein A-sepharose were incubated with GST or BRCT. Western blot analysis was then performed using antibodies against GST or FLAG. (B) GST-tagged fragments of DNA-PKcs encoding the amino acids indicated in the figure were purified from bacteria and left bound on the glutathione-agarose. These fragments were then incubated with purified His-tagged BRCA1 tandem BRCT domain protein fragment (BRCT). Following the incubation, the GST pull-downs were washed and western blot analysis was performed using anti-His and GST antibodies.