OSU-T315 targets intrinsic AKT and ERK signals cascades in CLL cells. (A) Lysate from Mec-1 and OSU-CLL cells treated with serial concentrations of OSU-T315 for 15 minutes are subjected to western blot analysis. (B) Lysate from primary CLL cells treated with OSU-T315 were subjected to analyze PDK1 (Ser241) and subsequent AKT (Thr308) activation. (C) In vitro kinase activity of class I PI3K is evaluated by the PI3 Kinase Activity/Inhibitor Assay Kit (Millipore) according to the instruction manual; 100 nM Wortmannin was applied as positive control. The biotinylated-PIP3 was set as 100%. The kinase reactions with vehicle or OSU-T315 were referenced to the biotinylated-PIP3 signal to have the relative percentage of inhibition. (D) RAS activity in 697 cells on treatments was measured by the Active Ras Detection Kit (Cell Signaling) according to the instruction manual. Guanosine triphosphate γS (positive control) and guanosine diphosphate (negative control) ensured the immunoprecipitation procedures worked properly, whereas insulin-like growth factor-1 served as a positive control to activate Ras. (E) Mec-1 and OSU-CLL cells pretreated with Okadaic acid (1 μM) were incubated with either OSU-T315 (4 μM) or OSU-03012 (5 μM), the PDK1 inhibitor, and the total lysate was subjected to immunoblotting to verify downstream signaling.