Figure 3. Gemcitabine and SN-38 induce Chk-1 phosphorylation and degradation.

Log-phase Ovcar-5 cells were treated for 24 h with diluent (0.1% DMSO, lanes 1 and 6), gemcitabine at 12.5, 25, 50 or 100 nM (lanes 2-5, respectively), or SN-38 at 2.5, 5, 10, 20 and 40 nM in the absence (lanes 7-11) or presence (lanes 12-17) of 50 nM gemcitabine. At the end of the incubation, whole cell lysates were prepared, subjected to SDS-PAGE, transferred to nitrocellulose, and probed with reagents that recognize phospho-Ser³⁴⁵-Chk1, total Chk1, topo I or, as control, β-actin. Because of the limited number of lanes on our electrophoresis apparatus, lanes 1-5 and 6-17 represent two different membranes from the same experiment that were probed simultaneously.