PKCη is required for mTORC1 activation by regulating mTOR localization.

<p>(A) Confocal microscopy images of immunofluorescence labeling of GFP and mTOR in PKCα-GFP-overexpressed HEK293 starved for 5 hours and then pretreated with 3 uM Bis for 1 hour followed by 100 nM PMA for 1 hour. Scale bar, 20 μm. (B) The level of phosphorylated and total S6K in HEK293 cells pretreated with 3 uM bisindolylmaleimide I (Bis) for 1 hour prior to treatment with 100 nM PMA for 1 hour. (C) Western blot analysis of phosphorylated and total S6K in HEK cells transfected with control, PKCη, PKCδ, PKCε, or PKCtheta siRNA and then treated with 100 nM PMA 5 hours after serum starvation. Bottom panels show efficiency of siRNAs on respective types of PKCs by protein level (PKCδ, PKCε) or mRNA (PKCη, PKCtheta). (D) Confocal microscope images of immunofluorescence labeling of mTOR and LAMP2 in HEK cells transfected with control or PKCη siRNA and treated with 100 nM PMA for 1 hour. Scale bar, 20 μm and 10 μm in enlarged images. Images are representative of three fields examined from three independent experiments. Western blots are representative of at least three independent experiments.