Differential effects of AD 198 and PEP005 on PKCδ subcellular translocation and cleavage. (A) Subcellular translocation of PKC isoforms. Mouse or human tumor B cells were cultured in the absence or presence of AD 198 or PEP005 for 10 minutes. Drug concentrations used: 1 μM AD 198 and 0.1 μM PEP005 for 105–8 cells; 4 μM AD 198 and 0.2 μM PEP005 for 8226 cells. Biochemical fractionation of cytosol, nuclei and membrane was performed as described in the Methods. Proteins were immunoblotted for PKCδ, phosphorylated-PKCδ (P-PKCδ), PKCϵ, and PKCα, followed by COXIV, HDAC1, and actin. Immunoblots of actin, HDAC1, and COX IV were used as loading control for proteins of cytosol, nuclei, or membrane (including mitochondria), respectively. Similar results were also obtained at 5 minutes or 30 minutes after treatment, and with another TRAF3-/- mouse B lymphoma cell line 27–9.5.3 and another human MM cell line 8226 (except barely detectable levels of PKCα in 8226 cells). (B) Phosphorylation and cleavage of PKCδ. Cells were cultured in the absence or presence of AD 198 of indicated concentration. Total cellular lysates were prepared at indicated time points, and then immunoblotted for phosphorylated-PKCδ (P-PKCδ), PKCδ, followed by actin. Similar results were also obtained with other cell lines examined. Results are representative of three independent experiments.