(E) Assay of GEF activity with fluorescence polarization measurements of 0.5 μM mantGDP-loaded CrARL3WT and 5 μM CrARL13B⋅GppNHpWT or CrArl13bE86R, to which 10 μM GppNHp (GTP analog) was added at the indicated time points. Only CrArl13bWT showed nucleotide exchange, as indicated by the drop in fluorescence polarization.