Optimization of biosensor expression in macrophages. Western blot analysis of A) original (non-optimized) or whitlow-modified Rac1 and Rac2 biosensors transiently expressed overnight. B) Tet-OFF inducible expression of original or whitlow-modified Rac1 and C) Rac2 biosensor. D) Tet-OFF induction of fully optimized, FACS-sorted Rac1, E) Rac2 and F) Cdc42 biosensors. BS = biosensor, Dox = doxycycline at 2 μg/mL, endg = endogenous, P = untransduced parental RAW/LR5 lysate, UT = untransfected.