Fig. 2: Dynorphin peptides directly bind to GPR139.

a Schematic of radioligand binding competition assays using cell membranes overexpressing GPR139. b Binding of 10 μM JNJ-63533054 and 40 μM dynorphin peptides to the membranes from HEK293T/17 cells transfected with GPR139 following co-incubation with 10 nM [³H]-JNJ-63533054. c Binding competition of [³H]-JNJ-63533054 with increasing concentration of dynorphin peptides. d Schematic of experimental design for detecting FITC-Dyn A13 binding to GPR139 by flow cytometry. e Flow cytometry histogram of cell population distribution after cell sorting. f Quantification of 1 μM FITC-Dyn A13 binding from (e). Median Fluorescence Intensity (MFI) was calculated. g Binding of 10 μM JNJ-63533054 and 40 μM Dyn A17 to the membranes from WT mouse brains in the presence of 10 nM [³H]-JNJ-63533054. h Binding competition of [³H] JNJ-63533054 with increasing concentration of Dyn A17. Data were normalized to specific binding of 10 nM [³H]-JNJ-63533054. i Quantification of the IC₅₀ values from the binding displacement curves in (h). Data are mean ± SEM from n = 3 independent experiments. In (b, g), data were analyzed by one-way ANOVA with Dunnett’s test or unpaired two-tailed t-test for (f, i). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.