Figure 4.

Active Cdc42 relieves the autoinhibition of FRLα localization. (A and B) Confocal images of RAW cells coexpressing mRFP, constitutively active Cdc42, and full-length FRLα-GFP (A) or mini-FRLα–GFP (B). (C–E) Confocal images of RAW cells expressing mRFP, full-length FRLα-GFP, and dominant-negative Cdc42 (C), constitutively active Rac1 (D), or constitutively active RhoA (E). (F) Confocal images of a cell expressing mRFP and the FRLα N terminus T126D-GFP mutant. (G and H) Confocal images of cells coexpressing N terminus–GFP and an mRFP fusion of WASP-GBD (G) or a mutant WASP-GBD (H) that does not bind Cdc42. For each transfection, 1 μg FRL construct was cotransfected with 4 μg GBD construct. (I) Membrane enrichment versus expression level for the indicated constructs. Each data point represents one transfected cell. (J) Coimmunoprecipitation experiments using lysates from 293T cells coexpressing myc-tagged Rho GTPase mutants and different FRLα-GFP constructs. Lysates were immunoprecipitated using anti-GFP or control IgG antibodies and blotted with anti-GFP or anti-myc antibodies. For each experiment (lanes a–c, d–f, g–i, j–l, m–o, and p–r), the lysate (starting material) is shown in the first lane followed by the material bound to control IgG beads or anti-GFP beads in the next two lanes. MW, molecular weight; SM, starting material.