Phosphorylation of FKHRL1 by EPO is absolutely dependent on PI3K activity.

EPO was removed from UT-7/EPO cells for 24 hours. The cells were pretreated with increasing concentrations of LY294002 (1-100 μmol/L) and then stimulated with EPO (10 U/mL) for 10 minutes. After solubilization, cell extracts 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). Anti-FKHRL1 antibody recognizes 2 bands; upper band (*) is the phosphorylated form, and the lower band (**), the unphosphorylated form.