Figure 3
Akt activity is required for PRL-3-mediated hyperactivation of mTOR signaling.
(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.
