Ligand-stimulated EPHA2 activation regulates Akt and Erk activation.

(A, B) Mutant EPHA2 proteins exhibit reduced activation of Akt and Erk by ephrin-A5. HEK293T cells were grown to confluence and serum-starved for 24 hours. 2 µg/mL cross-linked ephrin-A5-Fc was then added to the starvation media and cell lysates were immunoblotted with anti-phospho-Akt (Ser473) or anti-phospho-Erk (1/2), and then reprobed with anti-α-tubulin as a loading control. (C) Inactivation of EphA2 gene leads to reduction of Akt activity in mouse lenses. Each lens was prepared from 22 days old mice and extracted with lysis buffer. Total lens proteins were resolved by SDS-PAGE and western blot analysis was performed using indicated antibodies as described in the Materials and Methods. The blot was reprobed with anti-α-tubulin as a loading control.