Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 5
Specific knockdown of endogenous AKT isoforms. HEK293 cells were serum-starved for 24 hours and pretreated with either 5 μM AKTi, 20 nM rapamycin, or both for 30 minutes prior to stimulation with 10% serum for 20 minutes and harvesting into RLB. Endogenous AKT expression was knocked down, 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. Western blot signals were quantified by densitometry, normalised to loading and expressed as fold change over the serum-stimulated control. (a) Specificity of isoform-specific knockdown and their effects on total AKT expression were analysed by immunoblotting with isoform-specific and panAKT antibodies. (b) panAKT. . Error bars: mean ± SEM. (c) Total AKT activation levels were analysed by immunoblotting with phospho-Ser473 and phospho-Thr308 antibodies. (d) Phospho-Ser473. . Error bars: mean ± SEM. (e) Phospho-Thr308. , Error bars: mean ± SD.
