Quantification of MC4R Expression

The Nano-Glo^® HiBiT detection system (Promega, Mannheim, Germany) was used to quantify the cell surface and the total expression of MC4R [12 (link)]. Measurements were performed according to the manufacturer’s protocol. Two days after transfection, the medium was changed to 50 µL/well Opti-MEM without phenol red, and 50 µL of either HiBiT extracellular substrate (Promega, Mannheim, Germany) or HiBiT lytic substrate (Promega, Mannheim, Germany) was added. After orbital shaking for 3 min at 300 cycles/min and incubation at room temperature for 10 min, luminescence was measured using a Berthold Microplate Reader (Mithras LB 940, Berthold Technologies GmbH & Co. KG, Bad Wildbad, Germany). HEK-293 cells transfected with the empty vector pcDNA3 served as background control.