Intrinsic and Ric-8A-catalyzed GTPγS binding rates of the Gαi1 proteins used in this study.

<p>Intrinsic and Ric-8A-catalyzed kinetics of binding of GTPγS to wild-type Gαi1, W258A-Gαi1, NΔ25Gαi1, GαiCΔ9 and Gαi1-GαsC12 were measured using a fluorescence binding assay <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Thomas1" target="_blank">[12]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023197#pone.0023197-Higashijima1" target="_blank">[47]</a>. 400 µl of protein (1 µM) in the GDP bound form was equilibrated for 10–15 min at 25°C in a cuvette. A 10-fold excess of GTPγS was added and fluorescence at 340 nm upon excitation at 290 nm was monitored in the absence (open bars) or presence (filled bars) of Ric-8A (1 µM). Error bars represent +/− one standard deviation apparent first-order rate constants determined in three replicates.