Removal of endogenous ephrin-As from the cell surface potentiates EphA2 activation by soluble ephrin-A1 in <i>trans</i>.

<p>(A) SKBR3 and (B) MCF7 breast cancer cells were treated with PI-PLC for 4 hours and then stimulated with ephrin-A1 Fc. EphA2 immunoprecipitates were probed by immunoblotting for phosphotyrosine (PTyr) and reprobed for EphA2. Lysates probed with anti-ephrin-A1 antibody verify removal of ephrin-As by PI-PLC; β-tubulin verifies equal loading of the lanes. The Odyssey LI-COR system was used for detection and the color images were converted to greyscale with Photoshop. The histograms show the normalized data from 3 different experiments *p<0.05 and ***p<0.001 by one sample t test for the comparison of ephrin-A1 Fc-stimulated cells treated or not with PI-PLC. (C) SKBR3 cells were treated with PI-PLC as in A or with the broad-spectrum matrix metalloprotease inhibitor GM-6001 for 24 hours. Immunoprecipitates and lysates were probed as indicated.