Quantifying Mutant GPCR Expression

HEK293 stable cells and Neuro2a transiently transfected cells were treated with either 0.1% DMSO or Ipsen 5i (10⁻⁶ or 10⁻⁵ M) for 24 h at 37°C. On the day of experiment, cells were washed with ice-cold PBS-IH, detached, and precipitated by centrifugation at 500 × g for 5 min. Cells were then incubated with antibodies the same way as described above for confocal microscopy. For immunostaining of MC3R, cells were incubated with HA.11 antibody (Covance, Princeton, NJ, USA) at 1:100 dilutions and then stained with secondary antibody as described above. Cells were analyzed using a C6 Accuri Cytometer (Accuri Cytometers, Ann Arbor, MI, USA). Fluorescence of cells expressing the DMSO-treated empty vector (pcDNA3.1) was used for background staining. The expression of the mutants was calculated as percentage of DMSO-treated WT receptor expression using the following formula: [(mutant − pcDNA3.1)/(WT − pcDNA3.1) × 100%] (31 (link)).