3.11. PRESTO-Tango β-arrestin recruitment assay

The PRESTO-Tango (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation) assay was performed as recently described [27]. The Tango plasmids were a gift from Dr Bryan Roth (all from Addgene). HTLA cells (2.5 × 10⁵ per well) were seeded in a six-well plate and transfected with 1.5 µg of the Tango plasmids using Lipofectamine 3000 (ThermoScientific). The following day, transfected HTLA cells (1 × 10⁵ cells per well) were plated onto poly-l-lysine precoated 96-well microplates and allowed to attach to the plate surface for at least 4 h prior to treatment. Proteins used for treatment were prepared in twice the final concentration in culture media, added at a 1 : 1 vol/vol ratio and incubated overnight at 37°C, 5% CO₂ in a humidified environment. The following morning, media were removed from cell culture plates and replaced with a 100 µl 1 : 5 mixture of Bright-Glo (Promega) and 1× HBSS, 20 mM HEPES solution. Plates were then incubated at room temperature before measuring luminescence on a Biotek Synergy II plate reader.