Fig. 7. EphA1 inhibits the kinase activity of ILK. (A) Anti-FLAG (upper) and anti-ILK (lower) immunoblotting of endogenous ILK immunoprecipitated with anti-ILK antibody from the same set of cells as shown in Fig. 3D. (B) Mock and WT-EphA1–FLAG (WT) cells were plated onto FN-coated (1 µg/ml) dishes with or without immobilized ephrin-A1–Fc (1 µg/ml) for 15 minutes. Wortmannin (WM; 100 nM) was used for 30 minutes prior to the re-spreading of cells. ILK was immunoprecipitated and subjected to ILK kinase assay as described in the Materials and Methods. (C) Normalized kinase activities against the total amount of immunoprecipitated ILK (arrowhead) were quantified. Data on the right show means ± s.d. of three independent experiments. (D) HEK293 cells were transfected with control non-silencing RNA (Cont) or ILK siRNA (40 or 80 nM). Immunoblot analysis was performed 2 days after transfection. Cells transfected with control siRNA or ILK siRNA (80 nM) were plated on FN-coated (1 µg/ml) coverslips for 30 minutes. Cells were then stained with rhodamine-conjugated phalloidin (red) and DAPI (blue). Scale bar: 10 µm.