Figure 2.

Nucleotide exchange assays. 200 nM purified Fgd5-DH-PH-FYVE was incubated in the presence of 1 mM purified GST-Rac1 (a), Cdc42 (b) and RhoA (c) and fluorescent MANT-GTP to examine nucleotide exchange activity. 50 nM of the DH-PH domains from TrioN (a), Itsn1 (b) and Dbs (c) were used as controls which showed activation of Rac1, Cdc42 and RhoA, respectively. (d) GEF activity was calculated from the initial slopes of activation assays normalized to controls. (e) Comparison of the Fgd5-DH-PH-FYVE triple domain, Fgd5-DH-PH double domain and a double domain F803K mutant. GEF activities at 200 nM in the presence of 1 mM purified GST-Rac1. Representative results from single GEF assays are shown in the line graphs (a, b, c, e-left panel), while average activities calculated from four experiments are shown in the bar graphs (d, E-right panel). Error bars = s.e.m