Figure 2.

 Specificity of ceramide activity. (A) Relationship between PI(3)K inhibition by ceramide and apoptosis. Rat-1 Myc–ER cells were pretreated with 100 nm 4-HT for 60 min prior to 2-hr incubation with the indicated concentrations of C2 ceramide, then assayed for PI(3)K activity as in Fig. 1B (▪). Cells were pretreated similarly prior to incubation for 24 hr, harvested, and cell viability determined by trypan blue exclusion (•). (B) Cells were treated with 4-HT as in A, lysed, pooled, and equally aliquoted, followed by immunoprecipitation with anti-PI(3)K antibody, and treatment with the indicated amounts of C2 ceramide (solid bars), or dihydro-C2 ceramide (shaded bars), and assayed for PI(3)K activity. (10% FCS) Basal levels. Shown are representative results from three independent experiments.