Enforced activation of MEK/ERK attenuates adaphostin/MG-132 lethality. (A) Jurkat cells inducibly expressing a constitutively active MEK1 construct were incubated in medium in the presence or absence of 1 μM doxycycline for 30 hours, followed by exposure to 500 nM adaphostin and 250 nM MG-132 for 24 hours. At the end of 24 hours of drug exposure, apoptosis was monitored by annexin V/PI staining and flow cytometry. (B) Cells treated as in panel A were monitored for ROS generation after 16 hours of drug exposure. (C) Following 8 hours of drug exposure, Western blot analysis was employed to monitor the effect of drugs on expression of the indicated proteins. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. (D) U937 cells expressing a constitutively active MEK1 construct and stably transfected empty-vector cells were treated with 750 nM adaphostin plus or minus 250 nM MG-132. At the end of 24 hours, apoptotic cells were monitored. (E) U937 cells treated identically were assayed for ROS generation after 2 hours of drug treatment. (F) Following 8 hours of exposure of U937 cells to adaphostin and MG-132 as in panel D, Western blot analysis was employed to monitor effects on protein expression. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein. ^*Significantly less than values for cells exposed to drugs in the absence of doxycycline (A,B) or control pMM cells (D,E); P < .05.