Expression of HA tag, phospho-MEK, and phospho-ERK1/2. (A) Jurkat cells inducibly expressing a constitutively active, HA-tagged MEK vector under the control of a tetracycline-responsive promoter were exposed for 48 hours to 5 μM 17-AAG with or without 200 nM UCN-01 in the presence or absence of 2 μg/mL doxycycline. At the end of the incubation period, cell pellets were obtained, the cells lysed, and proteins separated by SDS-PAGE as described in “Materials and methods.” Western analysis was used to monitor expression of the HA tag, phospho-MEK, and phospho-ERK1/2. For each condition, lanes were loaded with 25 μg protein; blots were subsequently stripped and reprobed with antibodies to tubulin to ensure equivalent loading and transfer. The results of a representative experiment are shown; an additional study yielded equivalent results. (B) Following exposure to 17-AAG with or without UCN-01 for 48 hours in the presence or absence of doxycycline, the percentage of apoptotic cells was determined by morphologic assessment of Wright-Giemsa–stained cytospin preparations as described in “Materials and methods.” Values represent the means ± SD for 3 separate experiments performed in triplicate. **Significantly less than values obtained for cells exposed to UCN-01 plus 17-AAG in the absence of doxycycline; P < .005.