Prolume purple coelenterazine

Mini-G proteins coupled to Venus were from N. A. Lambert (Augusta University). Constructs were modified to replace Venus with Renilla (R) luciferase 8 (Rluc8). Renilla GFP-CAAX (prenylation CAAX box of KRas), tdRGFP-Rab5a and Rluc2-βARR2 were from M. Bouvier (Université de Montréal). HEK293T cells were transfected using PEI with hNK₁R (0.2 μg) and Rluc8-mGα_si/s/sq (0.1 μg) [30 (link),31 (link)]and either RGFP-CAAX (0.2 μg) for cell surface activation or tdRGFP-Rab5a (0.15 μg) for activation in early endosomes [32 (link)]. For EbBRET assays of recruitment of βARR2 to the plasma membrane or early endosomes, HEK293T cells were transfected with PEI with hNK₁R (0.2 μg) and Rluc2-βARR2 (0.1 μg) and RGFP-CAAX (0.2 μg) for plasma membrane recruitment or tdRGFP-Rab5a (0.15 μg) for early endosome recruitment [32 (link)]. For EbBRET assays, cells were washed with HBSS containing 10 mM Hepes (pH 7.4). Cells were incubated with Prolume Purple Coelenterazine (2.5 μM, 5 min, 37 °C; NanoLight Technology). EbBRET was recorded for 22.5 min in a Synergy Neo2 Microplate reader (BioTek) (acceptor filter: 515 ± 30 nm; donor filter: 410 ± 80 nm). Baseline was measured for 2.5 min and cells were challenged with SP (10 nM) or vehicle. ΔBRET represents the EbBRET signal in the presence of agonist subtracted by the EbBRET signal in the presence of vehicle.