ERK and FRS2alpha phosphorylation induced by FGF-21 in Klotho expressing cells. Klotho expressing HEK 293EBNA cells were serum starved for 16 hours and then stimulated with hFGF-23-His, FGF-23-Fc and FGF-b (positive control) for 10 minutes, respectively. Antibodies against pFRS2alpha, pERK1/2 and total ERK1/2 were used for immunoblotting. Lane 1: Mock (non-treated) Lane 2: Mock + hFGF23-Fc 1 ug/ml Lane 3: Mock + hFGF23-His 1 ug/ml Lane 4: Klotho + hFGF23-Fc 1 ug/ml Lane 5: Klotho + hFGF23-Fc 4 ug/ml Lane 6: Klotho + mCD137-Fc 1 ug/ml Lane 7: Klotho (non-treated) Lane 8: Klotho + 100ng/ml FGF-b