Fig. 7. Levels of p27^Kip1 are regulated by both MEK and PI 3-kinase pathways in Wt and Rb-deficient MEFs. Cyclin-dependent kinase activity was assessed following serum starvation (-) or following 16-20 hour restimulation with serum (+). For inhibitor experiments, cells were first pre-treated with inhibitors for 60 minutes prior to serum stimulation. Complexes were immunoprecipitated from 500 µg of cell lysates and cdk activity was assayed by in vitro kinase assay. (A) Endogenous cdk4 was immunoprecipitated and assayed using a C-terminal fragment of Rb as substrate. (B) Endogenous cyclin E was immunoprecipitated and assayed using Histone H1 as substrate. (C) Expression of p27^Kip1 was assessed by western blotting.