PEP005 actions are PKC-δ dependent. (A) Analysis by Western blotting of PKC-α,-β,-δ, and -ζ expression in 4 leukemic cell lines. β-actin was assessed as a loading control. (B) Expression of PKC-δ correlated with PEP005 responsiveness. PKC-δ expression was assessed by Western blotting in 4 leukemic cell lines and measured by densitometry. Data are expressed as PKC-δ expression relative to β-actin in the same sample. (C) Activation of PKC-δ by PEP005 (20 nM) and PMA (20 nM) was determined by measuring PKC-δ levels in the cytosolic (C), nuclear (N), and cell membrane (M) fractions of HL60 cells. (D) HL60 cells were incubated with 20 nM PEP005 for up to 9 hours and the presence of full-length (78-kDa) and the cleaved (40-kDa) fragment of PKC-δ was detected by Western blotting. (E) HL60 cells were incubated with 20 nM PEP005, 1 μM bisindolylmaleimide 1 (Bis-1), 20 nM Go6976, or PEP005 in combination with either Bis-1 or Go6976. Apoptosis was determined by caspase-3 activation. (F) HL60 cells were radiolabeled with ³²PO₄ prior to treatment with 20 nM PEP005 in the absence or presence of rottlerin and immunoprecipitation of caspase-3. An isotype-matched antibody (Irr) was used as a control. Blots in panels A, C, and D are representative of 3 separate experiments, and data in panels B and E are mean ± SD of 3 experiments.