Figure 2.

Cytokine activation of PKB and requirement for PI3K. (A) MC/9 cells starved of cytokine for 3–5 hr were stimulated with the indicated cytokines at concentrations that have been shown previously to induce maximal tyrosine phosphorylation and PI3K activity, for the indicated times. Also, some cells were pretreated with LY-294002 (25 μM) for 10 min and stimulated for 5 min with cytokine. Cells were pelleted and lysed in ice-cold solubilization buffer (see Materials and Methods). PKB was immunoprecipitated and its activity was measured in an in vitro kinase assay by using Crosstide as substrate. Individual experiments have been normalized to the percentage of stimulation induced by a 5-min treatment with IL-3, which was always performed concurrently. Typical maximum stimulations for IL-3, IL-4, and GM-CSF ranged between 8- and 12-fold above unstimulated samples, which generally were in the 2,000–3,000 cpm range. (B) Cells were prepared as above and pretreated with LY-294002 (25 μM) or vehicle alone for 10 min followed by stimulation with GM-CSF for the indicated times. PKB immunoprecipitation and kinase assay was performed as above. The results presented are from three experiments using duplicate samples, with error bars representing SEM.