Ectopic expression of constitutively active Raf-1 or a c-Jun dominant-negative mutant significantly protects cells from adaphostin/MG-132 induced lethality. (A) Jurkat cells inducibly expressing a constitutively active Raf-1 construct were incubated in medium in the presence or absence of 1 μM doxycycline for 30 hours, followed by exposure to 500 nM adaphostin plus 250 nM MG-132. After 24 hours of drug exposure, apoptotic cells were monitored by annexin V/PI staining and flow cytometry. (B) Alternatively, levels of ROS generation were determined after 16 hours of drug treatment. (C) Following 8 hours of drug exposure as in panel A, Western blot analysis was employed to monitor protein expression of Raf, phospho-ERK, and phospho-JNK. (D) U937 cells ectopically expressing a c-Jun dominant-negative construct (TAM67) or stably transfected empty-vector controls (pMM) were treated with 750 nM adaphostin plus 250 nM MG-132. At the end of 24 hours of drug exposure, apoptotic cells were monitored by annexin V/PI staining followed by flow cytometric analysis. (E) Following 8 hours of drug exposure, Western blot analysis was employed to monitor protein expression of phospho-c-Jun and phospho-ERK. Blots were stripped and reprobed with antitubulin antibodies to ensure equal loading and transfer of protein (20 μg each lane). ^*Significantly less than values for control cells; P < .05.