Tango Assay for GPR132 and ARRB2 Interaction

Tango assay was performed as described in our previous study [21 (link)]. CHO cells cultured in a 6-cm dish were co-transfected with GPR132-Tango, ARRB2-TEV, and TRE-Luc with a ratio of 2:1:1 by using polyethyleneimine (PEI). Transfected cells were then seeded into a 96-well plate at a density of 2 × 10⁴ cells per well. After 24 h, the medium was refreshed, and cells were incubated with compounds at indicated concentrations for 18 h. Subsequently, cells were lysed with Passive Lysis buffer (Promega, Madison, WI, USA) for 15 min at room temperature in a shaker at 70 rpm. The cell lysate was transferred to white 96-well plates, and the bioluminescence was determined immediately after the addition of luciferase substrate (Promega, Madison, WI, USA) with a Cytation^TM 5 Imaging Multi-Mode reader (BioTek, Winooski, VT).