Figure 6

Analyses of Rac1/IQGAP1/β-catenin complexes in EGFP-Rac1(V12)- or EGFP-Rac1(N17)-expressing PANC-1 cells. (A) Soluble (S100) and particulate (P100) fractions of EGFP-, EGFP-Rac1(V12)- and EGFP-Rac1(N17)-expressing PANC-1 cells were prepared from total cell lysates by centrifugation at 100000 × g. Subcellular localisation of Rac1, IQGAP1 and β-catenin was analysed in aliquots of 50 μg by Western blotting. To control for comparable loading β-actin was determined (lower panel). (B) Immunoprecipitation was performed using 300 μg of soluble (S100) or particulate, membrane-containing (P100) fractions of PANC-1 cells stably expressing EGFP, EGFP-Rac1(V12) or EGFP-Rac1(N17). After immunoprecipitation of IQGAP1 coprecipitated β-catenin and EGFP-Rac1 was determined by Western blotting. Detection of the immunoprecipitated IQGAP1 demonstrated equal amounts of protein in each precipitation. (C) Immunoprecipitation of β-catenin was performed in parallel experiments. Coprecipitated IQGAP1 and EGFP-Rac1 was determined by Western blotting and detection of the immunoprecipitated β-catenin verified equal amounts of protein. (D) The concentration of IQGAP1 in PANC-1 cells was reduced by transfection of two different siRNA oligonucleotides targeting IQGAP1. The amount of E-cadherin, β-catenin and IQGAP1 was demonstrated by Western blotting. Staining of β-actin served to demonstrate equal amounts of cell lysate. In each figure one representative blot out of three independent experiments is shown.