PK and bortezomib treatment triggers activation of c-Jun NH3-terminal kinase (JNK) and translocation of JNK from cytosol to mitochondria in MM.1S MM cells. (A) Cells were treated with PK (50 μM), bortezomib (2 nM), PK + bortezomib, PK + bortezomib + JNK inhibitor SP600125 (SP), or bortezomib (10 nM) alone for 24 hours. Protein lysates were immunoprecipitated with anti-JNK Ab. Immune complex kinase assays were performed by addition of 5 μg GST-Jun (2-100), (γ³²P) adenosine triphosphate (ATP), and incubation for 15 minutes at 30° C. The phosphorylated proteins were resolved by 10% SDS-PAGE and analyzed by autoradiography (top panel). Anti-JNK immunoprecipitates were also immunoblotted with anti-JNK Ab (bottom panel). Blots are representative of 3 independent experiments with similar results. (B) MM.1S cells were treated with indicated concentrations of PK + bortezomib for 24 hours. Cytosolic (Cyto) and mitochondrial (Mito) fractions were isolated and subjected to immunoblotting with anti-JNK (upper panel) or anti-Hsp60 (lower panel) Abs. Blots are representative of 2 independent experiments with similar results.