Fig. 8. Guanine-nucleotide binding to GST-Rac2^wt and GSTRac2^D57N, and association with upstream regulators of Rac. Guaninenucleotide binding to purified GST-Rac2^wt and GST-Rac2^D57N was performed as described in Materials and Methods. Each closed square represents GST-Rac2^wt and each open square represents GSTRac2^D57N. The data shown are a representative experiment of two such experiments. Each data point represents the mean±range of duplicate determinations. (A) Binding of [³H]GDP to GST-Rac2. GST-Rac2 was incubated for the indicated times with [³H]GDP. Data are expressed as picomoles of [³H]GDP bound per µg GST-Rac2. (B) Binding of [³⁵S]GTPS to GST-Rac2. GST-Rac2 was incubated for the indicated times with [³⁵S]GTPS. Data are expressed as picomoles of [³⁵S]GTPS bound per µg of GST-Rac2. (C) Dissociation of [³H]GDP from GST-Rac2 in the presence of an excess of unlabeled GDP. Prebound [³H]GDP was displaced by the addition of an excess of unlabeled GDP. The remaining bound [³H]GDP was measured at the indicated times. Data are expressed as the percentage of total [³H]GDP bound before the addition of unlabeled GDP. (D) Dissociation of [³H]GDP from GST-Rac2 in the presence of an excess of unlabeled GTPS. Prebound [³H]GDP was displaced by the addition of an excess of unlabeled GTPS. The remaining bound [³H]GDP was measured at the indicated times. Data are expressed as the percentage of total [³H]GDP bound before the addition of unlabeled GTPS. (E,F) COS7 cells were co-transfected with either RhoGDI or Myc-Tiam1 and the indicated expression constructs. An antibody to RhoGDI or to Myc was used to immunoprecipitate RhoGDI or Myc-Tiam1, respectively. The ability of Rac to immunoprecipitate with RhoGDI or Myc-Tiam1 is shown. Data are representative of between two and four experiments. (G,H) COS7 cells were transfected with indicated expression constructs and GST-PAKcrib binding was assessed. Data are representative of two such experiments.