Mutation of serine 65 on Bim confers resistance to UCN-01–mediated phosphorylation/degradation and sensitizes MM cells to UCN-01 treatment. (A) U266 cells were stably transfected with HA-tagged mouse wt and serine 65 mutant (S65A) Bim constructs or its empty vector (pCDNA3). The stable clones (1 for empty-vector control and wt Bim each and 2 for S65A mutants designated c19 and c32) were obtained by selection with G418. WB was performed to monitor expression of HA-tagged target proteins as well as phospho-ERK expression in these cells. (B) U266/wt Bim and U266/S65A Bim cells were exposed to 100 nM and 150 nM UCN-01 for 36 hours, after which ERK1/2 phosphorylation and HA-tagged Bim expression were monitored by WB. 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 (▼). Two additional studies yielded equivalent results. (C) Cells were treated as described for panel B, after which the percentage of apoptotic cells (annexin V⁺) was determined by annexin V–FITC staining and flow cytometry. Results represent the means (± SD) for 3 separate experiments performed in triplicate.*Significantly greater than the value for wt Bim-transfected cells with the same treatment (*P < .05 and **P < .01).