Activation of Akt by G-CSF treatment of BAF3 cells expressing the wild-type G-CSF receptor.

(A) Induction of Akt phosphorylation. Cells were incubated in serum-free medium for 6 hours and then stimulated with G-CSF (100 ng/mL) for 10 minutes with or without pretreatment for 15 minutes with wortmannin (WM) or Ly294 002 (LY). Akt phosphorylation was determined using a phospho-specific antibody that recognizes Akt only when phosphorylated on Serine 473 (upper panel). The membrane was reprobed with anti-Akt antibody (lower panel). (B) Activation of Akt kinase activity. Akt was immunoprecipitated from whole-cell extracts prepared from unstimulated or G-CSF–stimulated cells. The kinase activity of Akt was determined by in vitro kinase assay using histone H2B as a substrate (upper panel). The amounts of Akt kinase in each sample were determined by probing the membrane with anti-Akt antibody (lower panel).