FIG. 4.
Effect of LY294002 on alpha-toxin-induced generation of O2− and phosphorylation of PKCθ and PDK1. (A) Rabbit neutrophils (1.0 × 106 cells/ml) were preincubated with various concentrations of LY294002 at 37°C for 60 min. The cells were incubated with or without 1.0 μg/ml alpha-toxin at 37°C for the indicated periods. Symbols: control, ⋄; alpha-toxin, •; alpha-toxin plus 1.0 μM LY294002, ○; alpha-toxin plus 5.0 μM LY294002, ▴; alpha-toxin plus 10 μM LY294002, ▵. A typical result from one of five experiments is shown. (B) Rabbit neutrophils (1.0 × 106 cells/ml) treated with various concentrations of LY294002 were incubated with or without 1.0 μg/ml alpha-toxin at 37°C for 30 s. The extracted phospho-PKCθ and phospho-PDK1 were subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown. (C) Rabbit neutrophils (1.0 × 106 cells/ml) were preincubated with LY294002, U73122, and pertussis toxin, and the treated cells were incubated with or without alpha-toxin at 37°C for 10 min. In the case of pertussis toxin treatment, the cells were incubated at 37°C for 120 min. Intracellular DG levels were determined as described in Materials and Methods. Values represent means ± standard error for five to six experiments. *, P < 0.01, compared with production of DG induced by alpha-toxin alone. (D) Rabbit neutrophils (1.0 × 106 cells/ml) treated with various concentrations of U73122 and pertussis toxin were incubated with or without 1.0 μg/ml alpha-toxin at 37°C for 30 s. Phospho-PDK1 was subjected to SDS-PAGE and Western blotting using specific antibodies. A typical result from one of five experiments is shown.