Figure 3.

Perifosine inhibits Akt phosphorylation. (A) BCWM.1 cells were cultured with perifosine for 6 hours with doses that ranged from 2 to 20 μM. Whole cell lysates were subjecting to Western blotting using anti–p-Akt ser473, -Akt, –p-PDK1, –p-GSK3α/β, –p-S6R, and α-tubulin antibodies. (B,C) BCWM.1 cells were cultured with perifosine. Whole cell lysates were immunoprecipitated with anti-Akt antibody. Then the immunoprecipitated were washed and subjected to in vitro kinase assay according to manufacturer's protocol. Western blotting used -p-Akt, and fusion protein -p-GSK3α/β antibodies. (B) Dose effect of perifosine at 6 hours at doses that range from 5 to 20 μM. (C) Time-effect of perifosine at 10 μM for 2 to 6 hours.