Figure 2

EGFP-Rac1-induced differences in the amount of cell-cell adhesion protein concentration. (A) The amount of E-cadherin, α- and β-catenin in 30 μg of total cell lysate was analysed by Western blotting. Individual proteins were detected by using specific antibodies. Detection of β-actin served as control to document equal amounts of protein in each lane.(B+C) The amount of the E-cadherin/catenin complex was analysed by immunoprecipitation of β-catenin (B) or E-cadherin (C) from 300 μg of total cell lysate. Coprecipitated E-cadherin and α-catenin (B) or α-catenin and β-catenin (C), respectively, were identified by Western blotting. (D) Reverse transcription-PCR analysis was performed using total RNA isolated from PANC-1 cells stably expressing EGFP, EGFP-Rac1(N17) or EGFP-Rac1(V12). E-cadherin mRNA was amplified using specific primers. Amplification of β-actin cDNA served as control to document equal amounts of mRNA. (E) Association of the E-cadherin/catenin complex with the actin cytoskeleton was analysed using Triton X-100-fractionated cell extracts. Amounts of E-cadherin and β-catenin were analysed by Western blotting using 10 μg of Triton X-100-soluble and Triton X-100-insoluble fraction. (F) To investigate the influence of endogenous active Rac1 on the E-cadherin/catenin complex, PANC-1 cells were treated with 50 μM of the Rac1-inhibitor NSC23766 or stimulated for 5 min with PDGF AB (10 ng/ml). Active Rac1-GTP was precipitated from 800 μg of cell lysates by affinity precipitation assays (upper panel). To control for equal loading, aliquots of the samples were analysed in parallel (lower panel). The amount of E-cadherin/catenin complexes was analysed by detection of E-cadherin, which was coprecipitated with β-catenin from 500 μg of total cell lysates. (G) To investigate E-cadherin degradation in Rac1(V12)-expressing PANC-1 cells, cells were incubated with MG132 (1 μM) or E64 (1.5 μM) to suppress proteolytic degradation of proteins. The amount of E-cadherin was estimated by Western blotting using 30 μg of total protein lysates. Determination of β-actin served to control for comparable loading. In each figure one representative blot/agarose gel of three to four independent experiments is shown.