FIG. 6.

Requirement of KPC2 for p27 degradation. (A) Effect of KPC2 depletion by RNAi on p27 degradation. NIH 3T3 cells stably expressing KPC2 or EGFP shRNAs were synchronized in G₀ phase by contact inhibition and serum deprivation and then stimulated to reenter the cell cycle by replating at a density of ∼40% and incubation in complete medium for the indicated times. Cell lysates were then subjected to immunoblot analysis with antibodies to p27 or to HSP70 (control) (left panel). The intensity of the p27 bands was quantitated and plotted (right panel). (B) Complementation of impaired p27 degradation in NIH 3T3 cells expressing mouse KPC2 shRNA by introduction of human KPC2 derivatives. NIH 3T3 cells expressing KPC2 or EGFP shRNAs were infected with retroviral vectors for the indicated human KPC2 derivatives (tagged with the HA epitope) or with the corresponding empty retrovirus (Mock). The cells were then examined for p27 degradation during G₁ as in panel A. IB, immunoblot.