Fig. 4

Expression and activity of Akt isoforms. A The amount of the mRNA of AKT variants in three pancreatic and three lung adenocarcinoma cell lines and Colo-699 cells was analyzed by qPCR. Relative expression of each AKT variant was assessed in relation to the house keeping gene RPLP0. Three different cDNAs were used and analyzed in duplicate, mean ± SD is shown. Statistical analyses for pancreas and lung cell lines were done separately: One-way ANOVA (with Tukey’s multiple comparison tests), *** p ≤ 0.001, ** p ≤ 0.01; * p ≤ 0.05. B The expression of Akt isoforms in nine pancreatic, nine lung adenocarcinoma cell lines and Colo-699 cells was detected in RIPA cell lysates by Western blotting using Akt and Akt isoform-specific antibodies, β-actin served as loading control. Representative blots of n ≥ 3 independent experiments are shown. K-Ras mutation status is indicated behind the name: wild-type (−/−); heterozygous (−/+); homozygous (+/+); N*: N-Ras mutation. C-E Akt isoform-specific kinase activity. C Akt isoform-specific in vitro kinase assays were performed with serum-starved cells which were left untreated or treated with EGF (30 ng/ml; 5 min) or IGF1 (10 ng/ml; 5 min). Akt isoforms were precipitated with isoform-specific antibodies and 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. 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 are shown. D The bar graph shows the growth factor-stimulated Akt1–3 activity, as exemplary presented in C. The activity was evaluated by densitometric analyses, related to the amount of precipitated Akt1–3, and normalized to the untreated control cells set to 1. Mean ± SD (n = 3–5), one sample t-test, ** p ≤ 0.01; * p ≤ 0.05 is shown. E Representative Western blots of Akt isoform-specific kinase activity assays of serum-starved parental PANC-1 as well as EGFP- and EGFP-K-Ras(V12)-expressing PANC-1 cells. To control for unspecific binding of Akt [Control-IgG] a mouse-IgG2a antibody was used (n = 3)