Fig. 5. (A) Phosphorylation of - and ß-catenin after TGFß₁ treatment (10 ng/ml) for 30 minutes, 6 hours and 48 hours for BxPC-3 and additionally after 12 hours for PANC-1 cells was determined by immunoprecipitation of the individual proteins from isolated membrane fractions. Phosphorylated tyrosine was detected using a phosphotyrosine-specific antibody. Blots were restained with antibodies used for immunoprecipitation to document equal amounts of precipitated proteins. Representative blots out of three independent experiments are shown. (B) Phosphorylation of ß-catenin or (C) -catenin was examined after treatment of PANC-1 cells with TGFß₁, the PI3-kinase inhibitor LY294002 or the Src inhibitor PP1 (only for ß-catenin phosphorylation). Beta-catenin or -catenin was precipitated from 1 mg of PANC-1 RIPA-lysates treated with TGFß₁, TGFß₁ plus PP1 or TGFß₁ plus LY294002. Blots were restained for ß- or -catenin to document equal amounts of precipitated protein. (D) TGFß₁-induced ß-catenin phosphorylation was analysed in the presence of the farnesyltransferase inhibitor FTI 277 (2 µM) to inhibit Ras activity. In addition PANC-1 cells, which stably expressed EGFP/H-Ras N17, were analysed for ß-catenin phosphorylation in response to 10 ng/ml TGFß₁ or solvent. Beta-catenin immunoprecipitated from 1 mg of PANC-1 RIPA lysate was analysed regarding its phosphorylation by western blotting with a phospho-specific antibody. Equal amounts of ß-catenin were documented by restaining the blots with ß-catenin antibody. Representative blots are shown (n=3). (E) In addition to the pictures shown in Fig. 4D, immunolocalisation of p85 was performed in PANC-1 cells stably expressing EGFP/H-Ras N17 treated with TGFß₁ or solvent (–TGFß₁) for 3 hours and analysed with confocal laser microscopy. Bar, 20 µm.