Fig. 5.

CLLV-1 blocks AKT enzymatic activity but does not affect the AKT upstream kinases. (A-B) dHL-60 cells were preincubated with DMSO or CLLV-1 (0.03–1 μM) and then activated with or without fMLF (0.1 μM)/CB (1 μg/mL). (a) Phosphorylation of AKT upstream kinases, PDK1, mTORC2, and PI3K was determined by immunoblotting, using antibodies against the phosphorylated form and normalized to GAPDH. (b) PIP3 levels were assayed using anti-PIP3 antibodies by flow cytometry. (c) The active AKT proteins were immunoprecipitated using anti-phospho-AKT antibodies and treated with DMSO or CLLV-1 (0.3–3 μM) for 15 min at 30 °C. Subsequently, the GSK-3 fusion protein (AKT substrate) was added for another 30 min. The phospho-GSK-3 fusion protein was examined by immunoblotting. (d) The phosphorylation of the GSK-3 fusion protein was quantified and expressed as a percentage to represent AKT activity. All data are expressed as mean values ± SEM (n = 3); *p < .05 and ***p < .001 compared with the DMSO + AKT group (Student's t-test).