relative to untreated vector Supplementary Figure 2. Effects of PTEN expression and rapamycin treatment on phosphorylated S6 kinase. Effect of rapamycin on basal and EGF-stimulated S6 kinase phosphorylation in transfected PC-3 cells. Vector, wild-type and c.507delC mutant transient transfectants were incubated in serum-free medium with or without 100 nM rapamycin for 4 hours as indicated, then stimulated with 50 ng/ml EGF or vehicle control for 15 minutes. Harvested lysates were analyzed for phosphorylation of S6 kinase as described in Supplementary Figure 1. Densitometric quantification of pooled data from three experiments carried out in duplicate is shown in the bar graph as mean±SE, where phosphorylated S6 kinase is corrected for a-tubulin and data are expressed relative to untreated vector. Abbreviations: EGF, epidermal growth factor; phS6K, phosphorylated S6 kinase. EGF Rapamycin Vector PTEN c.507delC phS6K -tubulin + 1 2 3 4 5 6 relative to untreated vector