Enzastaurin specifically inhibits TPA-triggered phosphorylation and activation of PKC isoforms. (A) Enzastaurin inhibits TPA-induced phosphorylation of homologous PKC residues, but not PKC subcellular relocalization. Prior to TPA stimulation (200 nM, 20 minutes), MM.1S cells were treated with indicated concentrations of enzastaurin (3 hours) or left untreated. Immunoblots of cytosolic, membrane, and nuclear fractions were probed with indicated antibodies. The presence of gp130 in the membrane fraction and its absence in the cytosolic and nuclear fraction, as well as the presence of ERK-2 in the cytosolic and nucleolin in the nuclear fraction, served as controls for the purity of subcellular fractionation. (B-C) Enzastaurin specifically inhibits homologous Thr-514 and Ser-660 residue phosphorylation of PKC isoforms. MM.1S cells were treated with indicated concentrations of enzastaurin (3 hours), bisindolylmaleimide (BIM I, 2 μM), or left untreated. After stimulation with TPA (200 nM, 20 minutes), equal amounts of whole-cell lysates were immunoprecipitated with pPKC antibodies directed against the catalytic Thr-514 (B) or the regulatory Ser-660 (C) residue, and then immunoblotted with the indicated antibodies. IgH indicates immunoglobulin heavy chain. (d) Enzastaurin specifically inhibits conventional PKC isoform kinase activity. PKC isoform activity was determined using PKC immunoprecipitation kinase assays, as described in “Materials and methods.” (E-F) Enzastaurin specifically inhibits novel PKC isoform kinase activity. After stimulation with TPA (200 nM, 20 minutes), equal amounts of whole-cell lysates were immunoprecipitated with PKCδ (E) or PKCϵ (F) antibodies and immunoblotted with indicated antibodies. IP indicates immunoprecipitation; C1 (control 1), immunoprecipitation with protein A-Sepharose, lysis buffer, and specific antibody only; C2 (control 2), immunoprecipitation with protein A-Sepharose, whole-cell lysates, and preimmune rabbit serum.