RAD001-induced AKT S473 phosphorylation is dependent on rictor. Cell lines were transfected with 20 nmol/L control siRNA or siRNA against rictor or raptor for 72 h. Treatment within these 72 h, with vehicle control (DMSO) or 20 nmol/L RAD001, was according to the indicated time points. A, siRNA transfection efficiently down-regulates raptor and rictor protein expression in all lines tested. B, rictor down-regulation attenuates RAD001-induced AKT S473 phosphorylation in all lines. Raptor down-regulation induces basal AKT S473 phosphorylation and enhances the effects of RAD001. AKT T308 phosphorylation is independently regulated, showing attenuation of RAD001-induced effects by rictor down-regulation only in some of the cell lines tested. C, dephosphorylation of S6 on S235/236 as a control for RAD001-induced inhibition of mTORC1 signaling. AKT protein levels serve as loading controls.