Figure 5.

p120ctn induces Rac1 and Cdc42 activation. The level of active GTP-bound Rac1 (A) or GTP-bound Cdc42 (B) was measured in CHO cells transfected with either GFP or p120-GFP. Rac1 and Cdc42 activity assays were performed as described in Materials and Methods using a GST fusion protein derived from PAK (PBD) that selectively binds GTP-bound Rac1 and Cdc42. GST-PBD precipitations were immunoblotted with monoclonal antibodies recognizing Rac1 or Cdc42. Cell lysates were blotted with these antibodies to compare total levels of endogenous Rac1 and Cdc42 in transfected cells. In this representative experiment, p120-GFP induces a 2.4-fold activation of Rac1, the mean of four separate experiments being 1.9 ± 0.5. p120-GFP stimulates a 3.0-fold activation of Cdc42 in the representative experiment shown, the mean of three separate experiments being 3.1 ± 0.7. (C) The level of expression of p120-GFP and GFP in transfected cells was compared by blotting whole cell lysates with antibodies against p120ctn (left) or GFP (right). Endogenous p120ctn was detected as two bands, and p120-GFP was detected migrating above these. p120-GFP expression is usually two- to threefold higher than that of endogenous p120ctn.