GSK3 and Akt phosphorylation are PI3K dependent and phosphorylation of MAP kinase is Mek1 dependent in primary human erythroblasts.

Epo and SCF suppress GSK3 activity. Erythroblasts on day 6 of culture were deprived of growth factors for 2 hours before stimulation with Epo 20 U/mL, SCF 100 ng/mL, or both together as indicated, either with or without LY294002 (25 μM) or U0126 (10 μM) as indicated. Cell lysates were resolved using SDS-PAGE and probed on Western blot membranes using phosphospecific antibodies. The effect of PI3K blockade on GSK3 (A) and Akt phosphorylation (B) in a representative experiment of 4 performed is shown in addition to the effect of Mek1 blockade on MAP kinase phosphorylation (B). For kinase assays (C), erythroid progenitors on day 5 to 7 of culture were washed and incubated in medium without growth factors for 1 to 2 hours before stimulation with either Epo 20 U/mL or SCF 100 ng/mL. Kinase activity was determined at the times shown, either with or without preincubation with the PI3K inhibitor LY294002. The means and SEs are shown for 4 experiments (** denotes P < .05).