Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity
Figure 2
Comparison of AKT isoform-specific activation in vivo and activity in vitro. HEK293 cells were transfected with the pCDNA3 vector (control) or myrAKT isoforms, serum-starved for 24 hours, then stimulated with 10% serum for 20 minutes. (a) protein lysates (20–50 μg) were separated by SDS-PAGE, transferred onto PVDF membrane, and immunoblotted. Western blots are representative of experiments. Signals were quantified by densitometry using ImageJ 1.42 q (National Institutes of Health, USA), normalised to loading and expressed as fold change over myrAKT1 serum-starved samples. (b) panAKT. (c) HA-tag. (d) phospho-Ser473. (e) phospho-Thr308. (c–e) Serum-starved samples: , stimulated samples: . Error bars: mean ± SD. (f) protein lysates (20 μg) were incubated with the RPRAATF peptide substrate in the presence of [γ-32P]ATP at 30°C for 20 minutes to determine total AKT activity. Each sample was assayed in duplicate. Levels of AKT activity are represented as fold change over the serum-starved control sample. , Graph shows mean ± S.D. 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 (c and e) 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 (***).
