Dpn 1

Receptor mutagenesis was performed as previously described.^{47 (link)–49 (link), 54} The human WT N-terminal FLAG-tagged MC4R cDNA (Supplemental Figure 1A) was generously provided by Dr. Robert Mackenzie^{59 (link)} and was sub-cloned into the pBluescript plasmid (Stratagene) for subsequent mutagenesis. Site directed hMC4R mutagenesis was performed using a polymerase chain reaction (PCR) based strategy, using the Pfu turbo polymerase (Stratagene). Complementary sets of primers were designed containing nucleotide base pair changes resulting in the modified amino acids (Supplemental Figure 1B). Upon completion of the PCR reaction (95 ºC 30 s, 12 cycles of 95 ºC 30 s, 55 ºC 1 min, 68 ºC 9 min), the product was purified (Qiaquick PCR Purification Kit, Qiagen) and eluted in water. Subsequently, the sample was cut with Dpn1 (Invitrogen) to eliminate any methylated WT DNA, leaving only nicked circularized mutant DNA. The mutant hMC4R DNA was transformed into competent DH5α E. coli cells and single colonies were selected. The presence of the desired mutation was verified by DNA sequencing. The plasmid DNA containing the mutant was excised and sub-cloned into the HindIII/XbaI restrictions sites of the pCDNA₃ expression vector (Invitrogen). Complete FLAG-MC4R sequences were confirmed free of PCR nucleotide base errors by DNA sequencing (University of Florida sequencing core facilities).

Unless otherwise stated, reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Life Technologies. T4 polynucleotide kinase, T4 DNA ligase, molecular biology grade bovine serum albumin (BSA), and RNase inhibitor were purchased from New England Biolabs. γ-[³²P] ATP was purchased from Perkin-Elmer Life Sciences. The Avian Myeloblastosis Virus (AMV) reverse transcriptase, deoxynucleotide triphosphate (dNTP) mix and RQ1 RNase free DNase were purchased from Promega. Pfu Ultra II was purchased from Stratagene. Dpn 1 was purchased from Invitrogen. Quickchange XL II mutagenesis kit was purchased from Agilent Technologies. RNA oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility or purchased from GE Healthcare Dharmacon, Inc. or Sigma Aldrich. DNA oligonucleotides were purchased from Integrated DNA Technologies. Storage phosphor imaging plates from Molecular Dynamics were imaged using Molecular Dynamics 9400 Typhoon phosphorimager. Data were analyzed using Molecular Dynamics ImageQuant 5.2 software. Electrospray Ionization (ESI) mass spectrometry of oligonucleotide samples was carried out at the Campus Mass Spectrometry Facilities, UC Davis. Oligonucleotide masses were determined using Mongo Oligo Mass Calculator v2.06.