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Fig. 7. (A) Beta-catenin was immunoprecipitated from 1 mg of PANC-1 lysate of cells transfected with pEGFP, pEGFP-PTEN or pEGFP-PTEN
C, treated with 10 ng/ml TGFß1 or solvent for 6 hours and analysed for its tyrosine phosphorylation. The blot was restained for ß-catenin to demonstrate equal amounts of protein. (B) For an in vitro phosphatase assay, tyrosine phosphorylated ß-catenin was incubated for 30 minutes at 30°C with EGFP-PTEN-constructs, which were immunoprecipitated with anti-EGFP antibody. The amount of phosphorylated ß-catenin was analysed by western blotting. Restaining of the blots with anti-ß-catenin antibody revealed equal amounts of phosphatase substrate and restaining with anti-PTEN documents the presence of both EGFP-tagged PTEN proteins. Representative assays out of four independent experiments are shown. (C) Beta-catenin was immunoprecipitated from 1 mg of lysate from PANC-1 cells transfected with siRNA for PTEN and treated with TGFß1 (10 ng/ml) or solvent (two independent experiments each) or an unrelated control siRNA and analysed for its tyrosine phosphorylation. Restaining for ß-catenin is shown to document equal loading. PTEN protein expression was analysed by western blots stained for PTEN. Representative assays are shown (n=3). (D) Phosphorylation of Ser380 of PTEN, which was co-immunoprecipitated with ß-catenin, was analysed after TGFß1 treatment. Beta-catenin was immunoprecipitated from 2 mg of PANC-1 lysate treated with TGFß1 for 30 minutes or 6 hours. In addition PANC-1 cells were examined, which were pretreated with the farnesyltransferase inhibitor FTI 277 (2 µM) for 2 hours prior to addition of TGFß1 for 6 hours. Co-precipitated PTEN was analysed regarding the phosphorylation at Ser380 with a phospho-specific antibody. Restaining for ß-catenin documented equal amounts of precipitated protein. A representative blot out of three independent experiments is shown.
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