BRET assay was performed between MOR-Rluc8 and βARR2-Venus, MOR-YFP and βARR2-Rluc, or βARR2-RlucII and PAR2-YFP, as previously described (14, 64). Briefly, HEK cells were cotransfected with a DNA mix of 100 ng: 1 μg (donor-acceptor) (+100 ng of TRPV1, TRPA1, or pCDNA3 as control), using Lipofectamine (Invitrogen). At the next day of transfection, cells were plated in a white 96-well plate (BRAND). The agonist-induced BRET signal was calculated as the difference in BRET signal from cells treated with agonist or vehicle. For the dose-response BRET curve, cells were incubated with different concentrations of DAMGO for 15 min at 37°C and then washed and treated with the RLuc substrate coelenterazine h (5 μM) for 5 min before BRET measurement. BRET signal was measured in a Mithras LB940 (Berthold Technologies) and calculated as the ratio of YFP emission (530 nm) to the RLuc emission at (460 nm). BRET signal was expressed as net BRET, which is the difference between the signal from YFP/RLuc and the signal from RLuc alone.

For BRET assay between MOR-YFP and βARR2-Rluc, cells were transfected in 96-well plates using Lipofectamine 2000 (Invitrogen) with a DNA mix containing 100 ng of MOR-YFP plasmid [provided by S. Granier (Institut de Genomique Fonctionelle Montpellier)] and 2 ng of bARR2-Rluc plasmid [provided by M. G. Scott (Institut Cochin, Paris, France)] and 40 ng of TRPV1 plasmid [obtained from K. Talavera (Leuven, Belgium)] or empty vector. Twenty-four hours after transfection, BRET signal was measured using the pharmacological screening platform of the IGF, ARPEGE (www.arpege.cnrs.fr). Cells were washed twice with PBS and treated with coelanterazine-h (5 μM) for 5 min before stimulation with DAMGO (10 μM) or capsaicin (500 nM) or DAMGO and capsaicin. For bystander BRET, cells were transfected with a DNA mix of β-arr2-rLuc (100 ng), rGFP-NLS (100 ng), and TRPV1 or TRPA1 (100 ng).

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