FIG. 6.

CP-31398 stabilizes exogenous p53 by inhibiting its ubiquitination. (A) H460 cells were infected with Ad/p53 at multiplicities of infection (MOI) of 2.5, 5, and 10 for 12 h. CP-31398 (CP) was then added to the medium at a concentration of 15 μg/ml and incubated for 8 h. Cells were lysed for SDS-PAGE. p53 was probed with Ab-2. Also shown are p53^−/− HCT116 cells and p53^−/− MDM2^−/− MEF cells infected with Ad/p53 (MOI-5) and treated with CP-31398. C, control. (B) H460 cells were infected with Ad/p53 at a MOI of 5 (second panel). Twelve hours after the infection, CP-31398 was added and incubated for 1 h and then ALLN was added at a concentration of 50 μg/ml. Four hours later, cells were lysed for Western blotting by DO-1 against p53. Also shown are results performed in HCT116/p53^−/− cells (third panel) infected and treated as described for the H460 cells in the second panel.