Effects of different inhibitors on G-CSF–induced activation of Akt and Stats.

(A) BAF3 cells expressing the wild-type G-CSF receptor were unstimulated (lane 1) or stimulated with G-CSF for 10 minutes without (lane 2) or with preincubation with wortmannin (WM: 100 nM; lane 3), Ly294 002 (LY: 10 μM; lane 4), genistein (GN: 200 μM; lane 5), herbimycin A (HB: 1 μg/mL; lane 6), PP1 (10 μM; lane 7), bisindolylmaleimide (BM: 5 μM; lane 8) or cycloheximide (CHX: 30 μg/mL; lane 9). The preincubation times were 15 minutes except for herbimycin A (180 minutes). Whole-cell extracts were prepared and used for analysis of Akt phosphorylation by Western blotting. (B) The same extracts were used for the analysis of Stat5a activation by EMSA. (C) Peripheral blood neutrophils were left unstimulated or stimulated with G-CSF for 5 minutes following pretreatment with wortmannin or PP1 for 15 minutes as indicated. Whole-cell extracts were examined for Akt phosphorylation.