Ectopic expression of constitutively activate MEK1 promotes Bim_EL phosphorylation/degradation as well as diminishes the ability of PD98059 but not PD184352 to block UCN-01–induced Bim_EL phosphorylation. (A) RPMI8226 and U266 cells were stably transfected with a HA-tagged CA-MEK1 construct or its empty vector (pUSE). Western blot analysis was performed to confirm expression of HA-CA-MEK1 as well as to monitor phosphorylation/expression of ERK1/2 and Bim. (B) U266/pUSE and U266/CA-MEK1 cells were exposed to 100 nM UCN-01 plus or minus 50 μM PD98059 or 5 μM PD184352 for 40 hours, respectively, after which cells were lysed and subjected to Western blot analysis to assess ERK1/2 activation, Bim phosphorylation, and caspase-3 cleavage. Each lane was loaded with 20 μg protein; blots were stripped and reprobed with antitubulin antibody to ensure equal loading and transfer. Phosphorylated forms of Bim_EL are manifested by slowly migrating species (▼). CF indicates cleavage fragment. The results of representative experiments are shown; 2 additional studies yielded equivalent results.