Reduced cell surface expression of HT-hBRS3 after surrogate hBRS3 ligand stimulation (1 μM) was validated by FACS analysis.

After pulse-chase labeling of HT-hBRS3 at the cell surface with the HaloTag PEG-Biotin Ligand, cells were stimulated with surrogate hBRS3 ligands ([D-Phe6, ß-Ala11, Phe13, Nle14]-Bombesin (6–14); Phoenix Pharmaceuticals, Inc.). Following 3 h stimulation, fixed cells were stained with SA-APC (BioLegend) and analyzed by FACSVantage SE (BD Biosciences). Data are representative of two independent experiments. The red open histogram, no agonist stimulation; the blue open histogram, agonist stimulation (1 μM); the filled histogram, negative control. The horizontal bar indicates the FACS gate. Percentages of cells within the FACS gate are shown for each group. (EPS)