Fig. 5. Role of JNK activation in the PKC/Ras-mediated apoptotic process. In A, JNK activity, after chronic high-dose PMA treatment to down-regulate endogenous PKC activity, was assayed using GST-c-Jun (1–89) as the substrate in lysates containing equal amounts of total protein from cells with or without ectopic expression of v-ras, and phosphorylated c-Jun was visualized on a 12% SDS-PAGE gel (upper panel). The relative phosphorylation from each cell line was quantified using a phosphoimager (middle panel). A portion of the lysates from the same cells shown in the upper panel were immunoblotted with an anti-JNK Ab. In B, Jurkat/ras, Jurkat/Fasm/ras, Jurkat/FADDm/ras, or Jurkat/dn-FADD/ras cells, with or without the introduction of a dn-JNK1, wt-JNK1, or pGEX (mock), were treated with high-dose PMA for 48 h, and the percentage of DNA fragmentation in these cells was determined by flow cytometry. Bars, the SD over eight independent experiments. Ctrl, the untreated control cells ({square}); PMA, the cells treated with PMA (); PMA + dn-JNK1, the cells transfected with a dn-JNK1 before chronic high-dose PMA treatment ({blacksquare}); PMA + JNK1, the cells transfected with a JNK1 vector prior to chronic high-dose PMA treatment (dark gray column); PMA + pGEX, the cells transfected with a pGEX vector prior to chronic high-dose PMA treatment (light gray column).