. Effects of alanine substitutions to KB-752 binding, GDI activity, and attenuation of AC activation. (A) N-terminally biotinylated KB-752 mutant (W5A, F8A) or wildtype peptides were each immobilized to a density of ∼1000 response units (RUs) on separate streptavidin-coated flow cells and 25 μM of GDP-bound Gαs (“Analyte”) was injected simultaneously over all surfaces. (B) Purified Gαs or Gαi1 was incubated with the indicated KB-752 peptide and [35S]GTPγS binding was measured following incubation for 5 minutes at 20 °C. (C) cAMP accumulation over 10 minutes at 30 °C was measured from HEK293 cell membrane preparations stimulated with 10 μM GTPγS or 30 μM forskolin (FSK) in the absence (vehicle) or presence of 30 μM wildtype, palmitoylated KB-752 (wt) or the tryptophan-5 substituted version (W5A). *, p < 0.05; **, p < 0.01 compared to vehicle; one-way ANOVA followed by Dunnett's (n = 3).