Fig. 1.

Wnt-induced Rac1 activation and nuclear β-catenin translocation are dependent on p120-catenin. (A) SW-480 cells treated with control or Wnt3a-conditioned medium for the indicated time were lysed and active Rac1 was precipitated using a GST–PAK pull-down assay. Active Rac1 was detected by western blot (WB). All data are representative of at least five independent experiments. (B) The autoradiograms from experiments performed in triplicate as in A in SW-480 and HEK-293 cells were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nonstimulated cells. (C) SW-480 cells stably expressing scrambled or shRNA specific for p120-catenin (p120-cat) were treated with control or Wnt3a-conditioned medium for 2 hours. GST–PAK pull-down assays were performed with control and depleted cell lysates and active Rac1 was determined by WB. (D) Autoradiograms from five different experiments performed in triplicate as in C were quantified and the mean ± s.d. was obtained for each condition. Each value is presented relative to that obtained in nondepleted cells treated with control medium. (E,F) Cytosolic and nuclear lysates were obtained from control and p120-catenin-depleted SW-480 (E) or HEK-293 (F) cells treated with control or Wnt3a-conditioned medium for 15 hours. The nuclear fraction was separated from the cytosolic and membrane-associated fraction as detailed in Materials and Methods. β-catenin levels in each cellular fraction were analyzed by WB. Lamin-β1 was used as a nuclear marker and pyruvate kinase as a marker for the cytosolic-plus-membrane fraction.