ASK1 induces the activation of JNK and p38 MAPK and increases Bim expression. (A) Induction of ASK1 enhances activation of JNK and p38 MAPK. ASK1-inducible (ASK1) and control (TRE) H929 transfectants treated with Dox (1 μg/mL) were harvested on the days indicated, followed by immunoblotting of cell lysates with the indicated antibodies. Tubulin was used as an internal control. Each lowercase p indicates a phosphorylated protein. (B) Induction of ASK1 increases the expression of total Bim and enhances the phosphorylation of Bim_EL at residue S65. Lysates used in the experiments for panel A were used for immunoblotting with the indicated antibodies. (C) ASK1 is required for the Blimp-1 knockdown–mediated elevation of Bim level. H929 cells were transfected by electroporation with an siRNA that targeted ASK1 mRNA (ASK1i) or a nonspecific control siRNA (ctrli). After 24 hours, cells were transduced by a lentiviral vector to produce an shRNA against Blimp-1 (Blimp-1i) or a scrambled sequence (ctrli) for an additional 4 days. Cells were then harvested for lysate preparation, followed by immunoblotting with the indicated antibodies. (D) ASK1 is required for Blimp-1 knockdown–mediated apoptosis. H929 cells depleted of Blimp-1 or depleted of ASK1 and Blimp-1, as described in panel C, were subjected to annexin V staining followed by flow cytometric analysis.