Fig. 4. EphA3 juxtamembrane and activation loop tyrosines are required for sequential cell rounding and de-adhesion of EphA3 transfected 293T cells. 293T cells were transiently transfected either with pEGFP-actin (Clontech) alone (B, GFP-actin), or in addition to either wild-type (A, w/t) or mutant EphA3 with tyrosine to phenylalanine mutations at position 596 (Y596F), 602 (Y602F), 779 (Y779F), 596 and 602 (2xYF) or 596, 602 and 779 (3xYF) as indicated. Transfected cells on fibronectin-coated glass coverslips were stimulated with soluble clustered ephrinA5 Fc (1.5 µg/ml). Control samples were incubated with a 50-fold molar excess of soluble monomeric ephrin-A5 as inhibitor (B, w/t + Inhibitor) 30 minutes before addition of preclustered ephrin-A5. Cells were surveyed by confocal microscopy 10 minutes before and 50 minutes after the addition of ephrin-A5 Fc, and photos were taken at representative time periods selected. Bar, 20 µm.

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