Figure 5.

HCT-116 cells were pretreated with wortmannin 35.5 µM for two hours before treatment with DDP or taxol. At the end of DDP or taxol treatment cells were washed in PBS and medium containing wortmannin (35.5 µM) was added. Panel A: Extracts for DNA-PK activity were obtained 8 hours after treatment with and without wortmannin and processed as described in Materials and Methods. Values are the means±S.E. of pmolATP incorporated per minute per microgram protein in the biotinylated substrate in the absence (black columns) or in the presence of double-strand DNA (white columns). Panel B: Total cellular extracts were taken at 6 and 24 hours after treatment with wortmannin (lanes 2 and 5), with DDP (lanes 3 and 6), and with wortmannin and DDP (lanes 4 and 7). Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser15, Ser20 and p53 (DO-1). Panel C: Total cellular extracts were taken at 6 and 24 hours after treatment with wortmannin (lanes 2 and 5), with taxol (lanes 3 and 6), and with wortmannin and taxol (lanes 4 and 7). Blots were hybridized with antibodies recognizing p53 phosphorylated at Ser20 and p53 (DO-1).