Ephrin-A5–facilitated cell adhesion is accompanied by recruitment of focal adhesion components. (A) The distribution of EphA3 and of the focal adhesion proteins FAK, PTP-PEST, and paxillin was analyzed in LK63 cells adhering onto FN- or ephrin-A5-Fc–coated glass coverslips by optical sectioning on a confocal microscope. Alexa⁵⁴⁶ secondary antibody was used to detect α-EphA3 antibody; Alexa⁴⁸⁸ secondary antibodies were used to detect antibodies against all other proteins. Optical sections at the top of an LK63 cell and at the cell surface facing the bottom of the coverslip are illustrated for the paxillin/EphA3 costained sample. All other micrographs illustrate optical sections at the cell surface facing the coverslip. Scale bar represents 10 μm. (B) The recruitment of focal adhesion proteins to EphA3 was analyzed in α-EphA3 immunoprecipitates from LK63 cells adhering to FN-coated (−) or ephrin-A5-Fc–coated (+) surfaces, using antibodies against FAK, paxillin, and PTP-PEST, as indicated. The levels of each of the tested proteins in parallel cell lysates, as well as EphA3 levels in the IPs, are shown for comparison. (C) The expression of PTP-PEST was silenced by treating LK63 cells with PTP-PEST–specific siRNA; parallel cell cultures were transfected with cyclophilin control-siRNA for 48 hours. Whole cell lysates of stimulated (+) and control (−) cells were probed with antibodies as indicated. (D) To assess a potential role of PTP-PEST in LK63 adhesion, cells with silenced PTP-PEST expression and control siRNA-transfected cells were seeded onto FN- or ephrin-A5–coated glass coverslips; for confocal microscopic analysis, fixed cells were stained with antibodies/secondary antibodies against EphA3 (Alexa⁵⁴⁶, red) and PTP-PEST (Alexa⁴⁸⁸, green), and with Alexa⁶⁴⁷-phalloidin to mark filamentous actin. Representative images are shown. (E) The relative area of Eph clusters/cell, defined as ratio between the cellular footprints of EphA3 and of actin staining, was estimated from image raw data files using analySIS software. Mean and SE are shown, using data from 5 separate fields of view, each containing approximately 20 cells.