Fig. 5. Inhibition of Src kinase activity, Ras/MAP kinase signalling in PC12 cells by overexpression of APRO4. (A,B) PC12 cells transfected with a control empty vector or increasing amounts of APRO4, APRO4N, or APRO4C vectors were stimulated with FGF (25 ng/ml), and assayed for endogenous Src kinase activity. Proteins were immunoprecipitated from lysates containing 1 mg of total cellular protein with an excess of Src MAb 327 followed by in vitro kinase assay (A) or protein immunoblotting with anti-phospho-Y416 and anti-Src (B). Immunoblotting with Src antibody confirmed that equivalent amounts of Src were present in immunoprecipitates reactions. Data are representative of four independent experiments. (C) PC12 cells were co-transfected with 1 µg 5x-Gal4-TATA/luciferase, 200 ng Gal4-Elk1 (Elk/gal), 1 µg CMV-ßGal and the indicated expression plasmids, and either left unstimulated or stimulated with FGF (25 ng/ml) for 6 hours. Activation of Elk1 is reflected by luciferase activity normalized to ßGal activity and is described as fold decrease. Values are presented as the mean ± s.e.m. (error bars) of three independent experiments carried out in triplicate. Differences between two means with a P<0.05 were regarded as significant.