EPO induces phosphorylation of FKHRL1 protein in time- and dose-dependent fashions in UT-7/EPO cells.

EPO was removed from UT-7/EPO cells for 24 hours. The cells were then stimulated with EPO (10 U/mL) for the periods indicated (A), or with increasing concentrations of EPO (0.01-100 U/mL) for 10 minutes (B). 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.