Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 6
Dependence of AKT isoform-specific expression for signaling down cell proliferation, survival, and growth pathways. (a) HEK293 cells were serum-starved for 24 hours before pretreatment with 5 μM AKTi, 20 nM rapamycin or both for 30 minutes prior to stimulation with 10% serum 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. Western blots are representative of experiments. Intensity of western blot signals were quantified by densitometry, normalised to loading, and expressed as fold change over the serum-stimulated control. (b) phospho-FoxO1/3a (Thr24/32). . Error bars: mean ± SD. (c) phospho-PRAS40 (Thr246). . Error bars: mean ± SD.
