FKHRL1 is directly phosphorylated by EPO-activated Akt.

(A) EPO was removed from UT-7/EPO cells for 24 hours. Then the cells were stimulated with EPO (10 U/mL) for 10 and 20 minutes. After solubilization, cell lysates were immunoprecipitated with protein G-conjugated anti-Akt antibody. Then immunoprecipitates were subjected to an in vitro kinase assay. The kinase assays were carried out in the presence of 200 μmol/L ATP using GST-FKHRL1 fusion protein as a substrate (25 μg/mL). The reactions were incubated at 30°C for 30 minutes. Reaction products were resolved by 7.5% SDS-PAGE and immunoblotted with the antibodies directed against phospho-T32 (top panel) or phospho-S253 (middle panel). The blot was reprobed with anti-FKHRL1 antibody to confirm equal loading of protein (bottom panel).