Effects of dominant negative (DN) Jak mutants on the activation of Akt and Stat5.

(A) COS-7 cells were transfected with cDNAs encoding the wild-type G-CSF receptor and Stat5a only or were transfected together with cDNAs encoding the kinase inactive Jaks as indicated. Twenty hours after transfection, cells were starved for 4 hours prior to stimulation with G-CSF for 10 minutes. Whole-cell extracts were prepared and used for the analysis of Akt phosphorylation (upper panel). The membrane was reprobed with anti-Akt antibody (lower panel). (B) The same extracts were used for the analysis of Stat5a activation by EMSA using GRR probe. The complex that contains Stat5 is indicated with an arrow and labeled “GRR.”