Fig. 3. Functional expression of EphA1 in HEK293 cells. (A) The expression vector alone (pCMV-Tag4, Mock) or containing cDNA encoding WT- or KD (V638R)-EphA1–FLAG was stably transfected into HEK293 (293) cells. Expression levels of EphA1 and EphA2 in HEK293 Mock cells (lane 1), HEK293 WT-EphA1–FLAG cells (lane 2), K562 cells (lane 3) and EBC-1 cells (lane 4) are shown. Lysates from the indicated cells were analyzed by anti-EphA1, anti-EphA2 and anti-actin immunoblotting. (B) Mock, WT-EphA1–FLAG (WT) and KD-EphA1–FLAG (KD) cells were serum starved for 12 hours and stimulated with the indicated concentrations of ephrin-A1–Fc for 10 minutes, then lysed. EphA1 receptors were immunoprecipitated (IP) with anti-FLAG antibody and analyzed by immunoblotting (IB) with PY-20 (PY) and M2 (FLAG) antibody. (C) Immobilized ephrin-A1–Fc mediates adhesion of WT- and KD-EphA1–FLAG cells but not Mock cells. Cells were plated onto plastic culture plates or fibronectin (FN)-coated (1 µg/ml) 96-well plates with the indicated dosage of immobilized ephrin-A1–Fc (0-5 µg/ml) for 30 minutes. Adherent cells were fixed and stained with crystal violet. Dyes were extracted and measured at A590. (D) Mock, WT-EphA–FLAG and KD-EphA–FLAG cells were treated as in B (ephrin-A1–Fc at 1 µg/ml). Anti-FLAG immunoprecipitates were subjected to anti-FAK (upper) and anti-FLAG (lower) immunoblotting. (E) Mock, WT-EphA–FLAG and KD-EphA–FLAG cells were replated on FN-coated dishes with or without immobilized ephrin-A1–Fc (1 µg/ml) for 5 or 15 minutes. Then, cells were lysed and immunoprecipitated with anti-FAK antibodies for immunoblot analysis. Phosphorylated and total FAK in suspension (Sus) and in adhered cells were detected using anti-phosphotyrosine (PY) antibody followed by anti-FAK (FAK) antibody.