Measuring D3R Endocytosis in HEK-293 Cells

Endocytosis of D₃R was determined based on the hydrophilic properties of [³H]-sulpiride (Kim et al, 2001 (link); Guo et al, 2015 (link)). HEK-293 cells expressing D₃R were seeded (1.5 × 10⁵ cells/well) 1 d after transfection on 24-well plates. The following day, the cells were rinsed once and pre-incubated for 15 min with 0.5 ml pre-warmed serum-free medium containing 10 mM Hepes (pH 7.4) at 37°C. The cells were stimulated with 100 nM PMA or 1 μM isoproterenol for 30 min. They were then incubated with 250 μl of [³H]-sulpiride (7.2 nM) at 4°C for 150 min in the presence or absence of unlabeled competitive inhibitor (10 μM haloperidol). The cells were washed thrice with the same medium, and 1% SDS was added. Samples were mixed with 2 ml Lefko-Fluor scintillation fluid and counted on a liquid scintillation analyzer.