Fluorescence-based Rho GTPase GEF Assay

In vitro nucleotide exchange assay was performed as described (Kang et al., 2014 (link)) with modifications. Briefly, purified GST-Rho4 was incubated with 50-fold molar excess of mant-GDP (Invitrogen, Carlsbad, CA) in a nucleotide-loading buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM EDTA, and 2 mM DTT) for 25 min at 25°C. After termination of nucleotide loading with 20 mM MgCl₂, excess mant-GDP was removed by running through NAP™-5 gravity column (17-0853-01; GE Healthcare), and mant-GDP–loaded GST-Rho4 was switched into the GEF assay buffer (20 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM MgCl₂, 1% glycerol, and 1 mM DTT). A GEF assay was performed by mixing 40 μl of ∼2 μM mant-GDP-loaded Rho4 with 5 μl of GEF assay buffer containing 3FLAG-Gef3 (with different concentrations) or 3FLAG in the presence of 400 μM GDP. Fluorescence intensity was monitored at ∼20°C every 10 s using the Infinite M1000Pro plate reader (Tecan Group, Männedorf, Switzerland), with excitation at 360 nm and emission at 440 nm. Data were fitted with a monophasic exponential curves using y = m₁ − m₂ exp(−m₃x), where m₃ is the fluorescence decrease rate plotted in Figure 5, D and E (KaleidaGraph; Synergy Software, PA).