β-Arrestin recruitment assay.

The PRESTO-Tango system was utilized to screen ligand-receptor activation via the G protein–independent β-arrestin recruitment pathway (36). HTLA cells were plated onto 96-well plates (Corning) at 5 × 10⁴ cells/well in 100 μL medium and transfected with GPR75-Tango for 24 hours. Subsequently, the cells underwent a 2-hour serum-free DMEM starvation period. After starvation, the cells were rinsed and exposed to various reagents, incubating at 37°C with 5% CO₂ for 2 hours. Following this incubation, the treatment was removed, and fresh complete media (DMEM supplemented with 20% FBS) were replenished, allowing overnight incubation at 37°C with 5% CO₂. The next day, the plates were retrieved, and luminescence was measured using the Steady-Glo Luciferase Assay System (E2520, Promega) following the standard protocol.