Figure 4. Specific binding of [³H]-RvD5_{n-3 DPA} to human GPR101.

(A and B) Characterization of [10, 11, 13, 14 ³H]-RvD5_{n-3 DPA} ([³H]-RvD5_{n-3 DPA}). (A) RP-UV-HPLC chromatogram for RvD5_{n-3 DPA} and [³H]-RvD5_{n-3 DPA}. (B) RP-UV-HPLC chromatogram of [³H]-RvD5_{n-3 DPA} and online radioactivity monitoring. (C) GPR101-overexpressing CHO cells (0.5 × 10⁶ cells in 100 μL) were incubated with [³H]-RvD5_{n-3 DPA} at the indicated concentrations in the presence or absence of 10 μM RvD5_{n-3 DPA} (60 minutes at 4°C). Cell incubations were transferred to a vacuum manifold, unbound radioligand was removed, and activity was measured. Results represent the mean ± SEM (n = 4 from 2 distinct experiments). Inset shows a Scatchard plot. (D) To assess competition binding, GPR101-expressing CHO cells (0.5 × 10⁶ cells in 100 μL) were incubated with 3 nM [³H]-RvD5_{n-3 DPA} in the presence or absence of increasing concentrations of RvD5_{n-3 DPA} for 60 minutes at 4°C.