Exposure of U937 cells with or without BOC-D-fmk. U937 cells were exposed to 400 nM 17-AAG plus 75 nM UCN-01 for 10 hours in the presence or absence of 25 μM BOC-D-fmk, after which whole cell pellets were lysed, the proteins separated by SDS-PAGE, and Western analysis performed to monitor expression of Raf-1, total MEK, phospho-MEK, total ERK1/2, phospho-ERK1/2, phospho-JNK (A) or total Akt, phospho-Akt, phospho-GSKα/β, p34^cdc2, phospho-p34^cdc2, and phospho-p38 MAPK (B). Alternatively, cytosolic S-100 fractions were obtained as described in “Materials and methods,” the proteins separated as above, and expression of cytochrome c (cyto c), Smac/DIABLO, and AIF monitored by Western analysis. (C) Cells were treated with UCN-01 plus 17-AAG for 6 or 8 hours in the presence or absence of either BOC-D-fmk (25 μM), TPCK (20 μM), or MG-132 (500 nM), after which Western analysis was performed to monitor expression of Raf-1. For each condition, lanes were loaded with 25 μg protein; blots were subsequently stripped and reprobed with antibodies to actin to ensure equivalent loading and transfer. The results of a representative experiment are shown; 2 additional studies yielded equivalent results.