Fig. 5

Akt2 depletion upregulates Akt3 expression and activity. A Downregulation of individual Akt isoforms was performed by lentiviral transduction in PANC-1/K-Ras(V12) and PANC-1/EGFP pancreatic, as well as H23 lung carcinoma cells using Akt isoform-specific shRNAs. Expression of each Akt isoform was detected by Western blot analysis with Akt isoform-specific antibodies. B The bar graphs show the relative Akt isoform expression in relation to GAPDH or β-actin, normalized to the corresponding cell clone transduced with a scrambled shRNA [Scr] set to 1. Mean ± SD (n = 5–8, one sample t-test, **** p ≤ 0.0001, *** p ≤ 0.001, ** p ≤ 0.01; * p ≤ 0.05) is shown. C Akt isoform-specific kinase activity. Akt isoforms were precipitated with isoform-specific antibodies and the precipitate was incubated with recombinant GST-GSK3α/β as Akt substrate. To precipitate equal amounts of Akt isoforms the amount of lysate was adjusted according to the expression, except for the depleted isoform. Phosphorylation of GST-GSK3α/β and amounts of immunoprecipitated Akt isoforms were detected by Western blotting procedure using antibodies reactive to pGSK3 and Akt1/2/3. Representative blots (n = 3) are shown