Fig. 2

Detection of total AKT protein and phospho-protein by isoelectric focusing. Plots (d-f, h) show values after normalization to HSP70 levels analyzed in parallel in each sample. Symbols in plots: Red; KRAS mutated, green; BRAF mutated, blue; wild type (WT) with regard to KRAS and BRAF, black; unclear for KRAS and BRAF. a. Immunoblotting of selected tissue samples with antibodies against pAKT (AKT pS473) and total AKT protein. Blotting for β2 microglobulin (β2M) was used as a loading control. b. Representative electropherogram showing phosphorylated and non-phosphorylated AKT peaks. Blue and green lines indicate electropherograms of samples digested (green) or not (blue) with lambda phosphatase. Inset; electropherogram showing HSP70 run in parallel. c. Immunoblotting of HUVEC (±VEGF for 15 min) cell lysate with antibodies against pAKT and total AKT protein. Blotting for tubulin was used as to monitor equal loading. Control; without any incubation; Buffer; control lysate incubated with buffer, Phosphatase; lysate incubated with lambda phosphatase enzyme. d. Plot of total AKT peak areas (P1–P8) in the different samples. e. Plot of pAKT peak areas (present before but not after lambda phosphatase treatment; P1–P4 and P6). f. Plot of the ratio of pAKT/AKT peak areas. g. Representative electropherogram of p70S6 kinase expression. h. Plot of p70S6 kinase expression normalized to HSP70