Ligand binding assay using Gluc BRET

Ligand binding assay was performed as previously described [31]. HEK293A cells were transfected with Gluc-CXCR4 alone or in combination with Flag-LPA₁. One day after transfection, cells were dissociated with Accutase (StemCell Technologies) and centrifuged at 500g for 3 min. Cells were resuspended in DMEM without phenol red containing 5% FBS and seeded in a 96-well white plate at 2 × 10⁴ per well. The next day, cells were washed with HBSS and serum-starved for 2 h. In the saturation binding experiments, TZ14011-AF488 was treated in the presence or absence of 20 μM IT1t and incubated for 30 min at room temperature until reaching an equilibrium state. In the competition experiments, the medium was removed from each well, and 40 μl of TZ14011-AF488 was added, followed by a 30-min incubation at room temperature. Next, 10 μl of Coelenterazine native was added to achieve a final concentration of 5 μM, and the mixture was incubated for 5 min. Competing unlabeled CXCL12 (10 μl) was then manually added. Each plate was read using an LB942 Mithras microplate reader with a 480 nm filter for Gluc and a 540 nm filter for Alexa Fluor 488. Luminescence was measured at an integration time of 0.1 s per well for a duration of 30 min. The BRET ratio was calculated by dividing the Alexa Fluor 488 signal by the Gluc signal. The specific BRET ratio was calculated by subtracting the nonspecific BRET ratio from the TZ14011-AF488-induced BRET ratio. The ΔBRET ratio was calculated by subtracting the vehicle BRET ratio from the fluorescent ligand-induced BRET ratio.