Pcdna frt

Standard laboratory chemicals and reagents were obtained pure or at the highest available grade from commercial sources.^{24 (link)–28 (link),31 (link)} Gen Elute plasmid mini-prep kit, Dulbecco’s modified Eagle’s medium (DMEM), and cAMP kit were from Sigma (St. Louis, MO). AM3677 (Figure 1) was synthesized at the Center for Drug Discovery, Northeastern University (Boston, MA). CP55,940 and [³H]CP55,940 were obtained from the National Institute on Drug Abuse (Bethesda, MD). pRC/CMV-hCB1R was a generous gift from Dr. T.I. Bonner (National Institute of Mental Health, Bethesda, MD). Oligonucleotide primers were synthesized by Integrated DNA Technologies (Coralville, IA). The My Cycler PCR system was purchased from BioRad Laboratories (Hercules, CA). Pfu Turbo DNA polymerase was purchased as part of the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). One Shot Top10 Escherichia coli cells, Flp-In-293 cells, pcDNA/FRT, hygromycin, lipofectamine 2000, and pure link hipure plasmid filter midi-prep kit were purchased from Invitrogen (Carlsbad, CA). MinElute gel-extraction kits and the QIA Prep spin mini-prep kits were from Qiagen (Valencia, CA). Restriction endonucleases were purchased from New England Biolabs (Beverly, MA). Fetal bovine serum (FBS) and penicillin-streptomycin solution were purchased from GIBCO (Rockville, MD).