Figure 7: Effect of AKT expression on activation of the mTORC1 pathway. (a) HEK293 cells were serum-starved for 24 hours before treatment with 5 μM AKTi, 20 nM rapamycin or both for 30 minutes prior to stimulation with 10% FBS and harvested into RLB. Expression of endogenous AKT isoforms were knocked down in HEK293 cells, either individually or simultaneously, with 25 nM of siRNAs towards specific AKT isoforms, and harvested into RLB. siEGFP was used as the control. Protein lysates (20–25 μg) were separated by SDS-PAGE, transferred onto PVDF membrane, and immunoblotted. The arrow indicates the hyperphosphorylated band of phospho-4E-BP1. Western blots are representative of experiments. Intensity of western blot signals was quantified by densitometry, normalised to loading and expressed as fold change over the serum-stimulated control. (b) Phospho-rpS6 (Ser235/236). . Error bars: mean ± SD. (c) Phospho-rpS6 (Ser240/244). . Error bars: mean ± SEM.