Fig. 4. (A) Transwell migration assays were performed as described before. The inhibitors LY294002 (25 µM) or PD98059 (25 µM) were added to the upper compartment. TGFß₁ (20 ng/ml) or solvent was added to the lower chamber. The number of migrated cells was estimated after 48 hours of incubation. Mean values ±s.d. of one representative assay are shown out of three independent experiments. (B) The amount of PI3-kinase associated with the E-cadherin complex was analysed by co-immunoprecipitation. Beta catenin was precipitated from lysates of PANC-1 or BxPC-3 cells treated for 30 minutes, 6 hours or 48 hours with TGFß₁. The amount of co-precipitated p85 was analysed by western blotting. (C) The amount of p110 co-precipitated with ß-catenin was analysed in PANC-1 or BxPC-3 lysates. Beta-catenin was precipitated from 2 mg of PANC-1 or 1 mg of BxPC-3 NOP lysate treated with 10 ng/ml TGFß₁ or solvent. One representative blot out of three is shown. (D) Immunolocalisation of p85 was performed in PANC-1 cells treated with 10 ng/ml TGFß₁ or solvent (–TGFß₁) for 3 hours. P85 localisation was analysed with confocal laser microscopy. Bar, 20 µm.