Effect of 17-AAG and 17-AG on Chk1 and other polypeptides in HL-60 cells. (A) Log-phase HL-60 cells were treated for 6 hours (lanes 1 to 5) or 24 hours (lanes 6 to 10) with diluent (lanes 1 and 6), 100 nM 17-AAG (lanes 2 and 7), 300 nM 17-AAG (lanes 3 and 8), 1000 nM 17-AAG (lanes 4 and 9), or 3000 nM 17-AAG (lanes 5 and 10). After cells were washed in serum-free medium, whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and immunoblotted for Chk1 and Akt1. PARP, a well-recognized substrate of caspases 3, 7, and 9,^69-72  served as a marker of caspase activation. Chk2 served as a loading control. (B) Log-phase HL-60 cells were treated for 24 hours with diluent (lanes 1 and 6), 17-AAG (lanes 2 to 5), or 17-AG (lanes 7 to 10) at 30 nM (lanes 2 and 7), 100 nM (lanes 3 and 8), 300 nM (lanes 4 and 9), and 1000 nM (lanes 5 and 10). At the completion of the incubation, sample preparation and immunoblotting were performed as described in panel A.