Signal transduction events evoked by G-CSF treatment of rat cortical neurons. G-CSF activates STAT3, ERK, and PI3K/Akt pathways in primary cortical neurons. Western blots for phosphorylated proteins were stripped and reprobed with antibodies nonselective for phosphorylation. (A) STAT3 was rapidly but moderately phosphorylated (pSTAT3) 5 minutes after addition of G-CSF to the medium. Neuronal expression of the G-CSF receptor was also confirmed by Western blot analysis. Addition of the JAK2 inhibitor AG490 inhibited hyperphosphorylation of STAT3 5 minutes after addition of G-CSF. (B) Quantification of phosphorylation ratios from Western blots illustrates the moderate and transient but reproducible activation of STAT3 by G-CSF (data from 3 independent experiments). (C) G-CSF leads to increase of protein levels of the antiapoptotic STAT3 target Bcl-X_L in primary cortical neurons over 8 hours. (D) Induction of ERK1/2 (double band) and ERK5 by G-CSF. While ERK1/2 activation appears to be very transient (upper rows), ERK5 is induced for at least 60 minutes following G-CSF exposure (lower rows). (E) Stable induction of Akt phosphorylation upon addition of G-CSF to the medium (50 ng/ml) shown by immunoblotting with a Ser437 phosphorylation–specific antibody. In accordance with the known Akt activation pathway, the PDK kinase upstream of Akt was phosphorylated, and Akt phosphorylation could be blocked by preincubation of the neurons with the PI3K inhibitor LY294002. (F and G) Inhibition of PI3K by LY294002 diminishes G-CSF–mediated protection from apoptosis (measured by luminometric caspase-3/7 activity assay) in rat cortical neurons treated with staurosporine (F) or in the human neuroblastoma cell line SHSY-5Y, where cell death was elicited by camptothecin (G). Bars indicate mean relative protection (%) against the cell death stimulus ± SEM; values were normalized to the appropriate controls (n = 16 each).