Bg 782

HEK293 and SW480 human colon carcinoma cells were purchased from ATCC (Manassas, VA) and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 2 mM l-glutamine. Cells were plated at 3 million cells per 10 cm³ dish and transfected with 10 μg of cDNA of select GPCRs expressed in the pSNAP vector using 1 mg/mL polyethyleneimine (PEI). Twenty-four hours posttransfection, cells were trypsinized and replated onto six-well plates for SNAP-CHX-chase assays. Theoretical cDNA constructs were created with aPe-A Plasmid Editor v2.0 software. All pSNAP-GPCR cDNA constructs were created using Takara inFusion molecular cloning kits and sequenced for nucleotide accuracy by Eurofins. pSNAP vector cDNA and SNAP substrate BG-782 were obtained from New England Biolabs (Ipswich, MA). PageRuler Prestained NIR Protein, goat anti-rabbit DyLight 680 antibody, and ladders used for all NIR-PAGE analyses were from Thermo Fisher Scientific (Waltham, MA). Norepinephrine and phenylephrine were obtained from Sigma Inc. (St. Louis, MO)