Fig. 3. P63RhoGEF specifically displays in vitro exchange activity towards RhoA. (A) [³H]GDP nucleotide release assays on different recombinant Rho GTPases (0.4 µM) in absence (control) or in presence of GST-p63RhoGEF DH domain (2.8 µM), or GST-Dbl (1.2 µM). The exchange activity is expressed as the [³H]GDP remaining on the GTPases after 15 minutes of reaction. The experiment presented here is representative of at least three independent assays. Means and standard deviations are shown. (B) [³H]GDP nucleotide release assays on RhoA (0.4 µM) in absence (control) or in presence of GST-Dbl (1.2 µM), GST-p63RhoGEF DH (2.8 µM), or GST-p63RhoGEF L301E (2.8 µM). The exchange activity is represented as in A. (C) Time course study of guanine nucleotide exchange activity on the RhoA GTPase (0.4 µM) in absence () or in presence ([UNK]) of GST-p63RhoGEF DH (4 µM). The exchange activity is expressed as in A. (D) Kinetic of association of [³⁵S]GTPS to GDP-loaded RhoA (1 µM) in absence () or in presence ([UNK]) of p63RhoGEF DH (10 µM). Means and standard deviations are shown.