Supporting information for Mogil et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0730053100
Supporting Materials and Methods
MC1R Antagonist Synthesis and in Vitro Assays.
The following amino acids were coupled to the solid support: fluorenylmethoxycarbonyl (Fmoc)-Lys(Boc)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Nle-OH, Fmoc-D-Phe-OH, Fmoc-Trp(Boc)-OH, Fmoc-Asp(OtBu)-OH and Fmoc-Nle-OH. Each coupling reaction was achieved by using a 3-fold excess of the amino acid, 1-hydroxybenztriazole and 1,3-diisopropylcarbodiimide in dimethylformamide. The acetylation step was performed by treatment with 20% acetic anhydride in dimethylformamide for 25 min. The side-chain deprotection and peptide cleavage from the solid support was carried out by treatment with the mixture of 90% trifluoroacetic acid, 2.5% 1,2-ethanedithiol, 2.5% anisole, 2.5% thioanisole, and 2.5% water for 3 h. The crude peptide was purified by reverse-phase HPLC using a semipreparative Vydac column C18 (218TP, 10 mm ID × 250 mm length) and UV detection at 254 nm. The purity was found to be > 96% by analytic HPLC and electrospray ionization MS.Human Genotyping.
Patient DNA (PureGene system, Gentra Systems, Minneapolis) was amplified with standard conditions by using the following three sets of primers covering most of MC1R: MC1R-1F, 5'-TGCAGCAGCTGGACAATG-3' + MC1R-1R, 5'-ATGTGGACGTACAGCACGG-3' (bases 338–630 of the cDNA; Genbank accession no. X65634); MC1R-2F, 5'-TGCATCTCACTCATCGTCC-3' + MCIR-2R, 5'-ATATCACCACCTCCCTCTGCC-3' (bases 776–1004); MC1R-3F, 5'-CCTGGCAGCACCATGAACTA-3' + MC1R-3R, 5'-AGAGGCTGGACAGCATGGAG-3' (bases –97 through 397). MC1R-1 and MC1R-3 products were cycle sequenced (Applied Biosystems BigDye Terminator kit and ABI 310 machine), using the PCR primers plus MC1R-3a (5'-TGCCTGGAGGTGTCCATCT-3') and MC1R-3b (5'-GTTCTCCACCAAGCTCACCA-3') as internal sequencing primers. The MC1R-2 PCR products were digested with α-TaqI restriction enzyme, followed by native PAGE, to detect the D294H mutation via loss of digestion.