Cell Permeabilization and BRET Assay

Cells were washed with DPBS, harvested by trituration, and transferred to opaque black or white 96-well plates containing diluted drug solutions. For assays with nucleotide-free heterotrimers (Fig. 4), cells were washed with permeabilization buffer containing 140 mm KCl, 10 mm NaCl, 1 mm MgCl₂, 0.1 mm K-EGTA, 20 mm NaHEPES (pH 7.2), harvested by trituration, and permeabilized in the same buffer containing 10 μg ml⁻¹ high purity digitonin (EMD Millipore, Burlington, MA). Measurements were made from permeabilized cells supplemented either with GDP (0.5 mm) or apyrase (2 units ml⁻¹; Sigma) and agonist. BRET and luminescence measurements were made using a Mithras LB940 photon-counting plate reader (Berthold Technologies GmbH, Bad Wildbad, Germany). Coelenterazine h (5 μm; Nanolight, Pinetop, AZ) or furimazine (Nano-Glo; 1:1000, Promega Corp.) were added to all wells immediately prior to making measurements with Rluc8 and Nluc (or Nluc fragments), respectively. Raw BRET signals were calculated as the emission intensity at 520–545 nm divided by the emission intensity at 475–495 nm. Net BRET was this ratio minus the same ratio measured from cells expressing only the BRET donor. NES–venus–mG fluorescence in Fig. 2 was measured using a Guava 6HT/2L flow cytometer (excitation 488 nm, detection 525/30 nm) and reported as average fluorescence from all positive cells.