Figure 2.

Activation of PI3-K and Akt by hGM-CSF in BA/F-wild cells. Activation of PI3-K and Akt in BA/F-wild in the presence or absence of wortmannin (A and B) or in BA/F-wild and BA/F-Fall cells (C and D) is shown. BA/F-wild cells were depleted of mIL-3 for 5 h and restimulated with hGM-CSF for 5 min. For PI3-K assay, immunoprecipitation was done using anti-phosphotyrosine antibody (4G10), and immunoprecipitates were subjected to kinase assay using phosphatidylinositol and phosphatidylserine as substrates in the presence of [γ-³²P]ATP. Products were separated by TLC plates. Arrows indicate applied origin and kinase reaction product phosphatidylinositol phosphate (PIP). The bar graph under the TLC pattern indicates the radioactivity intensity of products calculated by image analyzer (Fuji BAS-2000). Akt activity was analyzed by Western blotting of total cell lysates using anti-phospho-Akt antibody. The upper panel is the blotting pattern of anti phospho-Akt antibody, and the lower panel is the reblotting pattern of the same filter using anti Akt antibody.