Ephrin-A5–induced LK63 cell adhesion and spreading result from PTP–down-modulated EphA3 kinase activity. (A) The dose response (top panels) and time dependence (bottom panels) of sodium pervanadate (vanadate) induced EphA3 phosphorylation in EphA3/HEK293T and LK63 cells were analyzed in α-PY Western blots of EphA3 IPs. For comparison, the ephrin-A5-Fc–induced phosphorylation in EphA3/HEK293 cells is shown. Short (S) and long (L) exposures of the dose-response panels are shown to emphasize significantly different phosphorylation levels. (B) Adhesion and spreading of LK63 cells on ephrin-A5 surfaces is abrogated in the presence of 1 mM sodium pervanadate: The actin cytoskeleton of LK63 cells adhering to ephrin-A5 surfaces was imaged by confocal microscopy. A 2.7-fold magnification reveals filopodia-like cell processes in the adherent LK63 cells (white arrows), and membrane blebbing in the pervanadate-treated cells exposed to the ephrin-A5 surface. Scale bar represent 20 μm. (C) The number of cells with round (contracted) morphology (■) and of spread cells with filopodia-like extensions () was counted by a blinded observer in a minimum of 3 microscopic fields. Mean and SE were estimated from data representative of 2 independent experiments. (D) The effect of the sodium pervanadate treatment on LK63 cell viability was assessed by trypan-blue exclusion. (E) Confocal microscopic images of LK63 cells seeded onto ephrin-A5/FN–coated surfaces and probed with Alexa⁴⁸⁸-phalloidin and anti–PY-EphA3/Alexa⁵⁴⁶ secondary antibodies. Cells in the lower panels were treated with 1 mM sodium pervanadate as indicated. Scale bars represent 20 μm.