Quantifying Exogenous MC4R Expression

HEK293T cells were transiently transfected with hMC4R or LmMC4R plasmid with N-terminal c-myc tag. Forty-eight hours after transfection, cells were incubated with mouse anti-myc 9E10 monoclonal antibody (Developmental Studies Hybridoma Bank, The University of Iowa, Iowa City, IA, USA) diluted 1:40 for 1 h. Cells were then washed and incubated with Alexa Fluor 488-labeled goat anti-mouse antibody (Invitrogen, Grand Island, NY, USA) diluted 1:2,000 for 1 h. The C6 Accuri Cytometer (Accuri Cytometers, Ann Arbor, MI, USA) was used for analysis. Fluorescence of cells expressing the empty vector (pcDNA3.1) was used for background staining. The expression of the LmMC4R was calculated as percentage of hMC4R expression using the following formula: [(LmMC4R – pcDNA3.1)/(hMC4R – pcDNA3.1) ×100%] (42 (link)).