Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 3
Differential isoform-specific signaling to direct AKT substrates in vivo. (a) Protein lysates (20–50 μg) generated from HEK293 cells transfected with the pCDNA3 vector (control), or overexpressing HA-tagged myrAKT isoforms were resolved by SDS-PAGE, transferred onto membrane, and immunoblotted. Western blots are representative of experiments. Signals from serum-starved samples were quantified by densitometry using ImageJ 1.42q (National Institutes of Health, USA), normalised to loading, and expressed as fold change over myrAKT1 serum-starved samples. (b) phospho-GSK3 (Ser21). . Error bars: mean ± SEM. (c) phospho-GSK3β (Ser9). . Error bars: mean ± SEM. (d) phospho-FoxO1/3a (Thr24/32). . Error bars: mean ± SEM. (e) phospho-FoxO1 (Ser256). . Error bars: mean ± SEM. (f) phosph-PRAS40 (Thr246). . Error bars: mean ± SD. Statistical analysis was performed using the paired t-test (GraphPad Prism version 5.0, GraphPad Software, San Diego, Calif, USA). Paired t-test was not calculated between serum-starved control and myrAKT1 for (d) as the fold difference was the same for all blots quantified. values >0.05 are not significant, values 0.01 to 0.05 (*), values 0.001 to 0.01 (**), and values < 0.001 (***).
