Pcdna3 expression vector

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).

Rat Mgat5 cDNA (Shoreibah et al. 1993 (link)) was subcloned into the EcoRI site of the pcDNA3 expression vector (Invitrogen, San Diego, CA). Lec4 cells were plated on 35 mm cell culture dishes and grown in α-MEM medium supplemented with 10 % fetal calf serum. Plasmid DNA (1 μg per 35 mm culture dish) was transfected into Lec4 cells, using Fugene™ 6 transfection reagent (Boehringer Mannheim, Germany) according to the manufacturer's protocol. Following transfection, clonal cell lines were established by selection for G418 resistance and tested for cell surface expression of β1,6 GlcNAc-branched N-glycans using digoxigenin-conjugated leukoagglutinating Phaseolus vulgaris lectin (dig L-PHA; Boehringer Mannheim, Germany) as described below. The positive clonal cell lines were designated Lec4 GnTV-N5, Lec4 GnTV-N10, and Lec4GnTVN30. Lec4 cells were mock-transfected with the pcDNA3 expression vector as described above, and clonal cell lines were established and designated Lec4 pcDNA3.