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. Author manuscript; available in PMC: 2010 Jul 7.
Published in final edited form as: Cancer Cell. 2009 Jul 7;16(1):9–20. doi: 10.1016/j.ccr.2009.04.009

Figure 1. EphA2 possesses both ligand-independent and ligand-dependent functions in regulating growth factor-induced chemotaxis.

Figure 1

A–B. Ectopic overexpression of EphA2 enhances serum-induced chemotaxis in a ligand-independent manner. U373 cells were infected with retroviral vector expressing EphA2. Cells were stimulated with ephrin-A1-Fc or Fc, and lysates were blotted for active (p-EphA/B) or total EphA2 (A). The infected cells were subjected to Boyden chamber cell migration assay (B) with 5% FBS in lower chamber as chemoattractant. Cell numbers from 6 random fields were counted. Numbers represent mean ± S.D. ***, p<0.001; ###, p<0.001.

C–F. Downregulation of EphA2 by shRNA reduces serum-induced chemotaxis of U373 and PC-3M cells. EphA2 shRNA or control GFP shRNA was introduced into U373 (C–D) and PC-3M cells (E–F) via Lentiviral infection. Stable cell lines were subjected to immunoblot (C, E) or chemotactic cell migration assay (D, F) as described for A and B. Numbers represent mean ± S.D. **, p<0.01; ***, p<0.001; ##, p< 0.01; ###, p<0.001.

G–H. Serum stimulation results in S/T phosphorylation of EphA2 downstream from Akt activation, which is inhibited by ephrin-A1 co-stimulation. Serum-starved cells were scratch-wounded and stimulated with FBS for the indicated times in absence and presence of ephrin-A1-Fc. Total cell lysates (G) or EphA kinase precipitates (H) were subjected to immunoblot with the indicated antibodies.

I. Serum-induced EphA2 phosphorylation at Akt substrate sites is abolished by pretreating cells with LY294002. Experiments were performed as described in G–H except that 10 μM of LY294002 was added to the cells 1 hour prior to serum stimulation.

J. Serum-induced EphA2 phosphorylation at Akt substrate sites is inhibited by DN-Akt. U373 cells that stably expressed DN-Akt or control vector were stimulated and analyzed as described in G–H.

K. EphA2 kinase activation by ephrin-A1 inhibits leading edge localization of phosphorylated Akt. Cells were starved and wounded as descried above. After stimulation for 10 min with serum in the absence and presence of ephrin-A1-Fc, cells were fixed and stained as described in the methods. Scale bar, 25 μm.