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. 2015 Nov 24;5:17046. doi: 10.1038/srep17046

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Copyright © 2015, Macmillan Publishers Limited

This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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(a) Akt in HCT116 cells stably expressing EGFP (Vec) or EGFP-PRL-3 (PRL-3) were transiently depleted using scrambled small interfering RNA (siRNA; siCon) or AKT-targeting siRNA (siAKT) and cultured for 48 h before analysis of Akt-mTOR pathway activity. The ratio of phosphorylated/total band densities for Akt, 4E-BP1, and p70S6K were calculated and normalized to their cognate phosphorylated/total protein ratio in lane 3. (b) Akt in HCT116 Vec or PRL-3 cells was inhibited for 30 min using Akt inhibitor VIII (AktiVIII) before analysis of Akt-mTOR pathway activity as in (a). The ratio of phosphorylated/total band densities for Akt, 4E-BP1, and p70S6K were calculated and normalized to their cognate phosphorylated/total protein ratio in lane 3. Full immunoblots for the cropped images presented here are provided in the Supplementary Information.