Supplementary Figure 10: Effects of p.Val29Leu mutant on ARHGEF26 protein nucleotide-exchange activity.

Evaluation of wild-type and Leu29 ARHGEF26 nucleotide-exchange activity. Full-length, N-terminally His-SUMO-tagged wild-type and Leu29 ARHGEF26 and full-length RhoG were expressed in E. coli. The nucleotide-exchange assay was prepared with equal amounts of recombinant ARHGEF26 WT (blue) and ARHGEF26 Leu29 (red) in reaction buffer containing MANT-GTP. Just prior to reading, recombinant RhoG protein, preloaded with GDP, was added to the reaction buffer at a final concentration of 0.4 μM. MANT-GTP fluorescence was monitored for 60 min on a SpectraMax M2 at 37 ºC using an excitation wavelength of 280 nm and an emission wavelength of 440 nm with a cutoff at 435 nm. No significant difference in nucleotide-exchange activity was observed between ARHGEF26 WT (blue) and ARHGEF26 Leu29 (red) in the presence of RhoG.