PRESTO-Tango Assay for GPRC5C

PRESTO-Tango assay was performed according to the protocol in Kroeze et al.^{36 (link)}, with slight modifications. Briefly, HTLA cells were cultured in 10% FBS DMEM with 2 μg ml⁻¹ puromycin and 100 μg ml⁻¹ hygromycin B. Cells were transfected with 30 μg ml⁻¹ of the GPRC5C-Tango plasmids using JetPrime. After 24 h of transfection, cells were plated at 60,000–80,000 cells per well into a 96-well plate. Then, 3.5× concentration of the drug was prepared in 20 mM HEPES and 1× HBSS, pH 7.4, and 40 μl of the drug population was added to each well. After 24 h of incubation, cells were washed with PBS and transferred into a 96-well plate (Greiner) with 40 μl of Bright-Glo solution (Promega). Following a 5-min incubation at RT, each well was measured for 5 s on a Centro LB 963 Microplate Luminometer (Berthold).