Supporting information for Tanka et al. (2003) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0837789100
Fig. 7.
Mutational analysis of the CTG translation initiation codon of mouse prepro-NPW (neuropeptide W) cDNA. (A) 5'-flanking region of human and mouse prepro-NPW cDNA. Putative translation initiation codon is marked with asterisks. Secretory signal sequences predicted by the SignalP server (1) are marked by underlines. Arrows indicate purines (G) at positions –3 and +4. Codons for mature peptides are boxed. (B) Mouse prepro-NPW cDNAs with designated start codon and C-terminal FLAG tag were synthesized, and transfected into Chinese hamster ovary (CHO) cells. Cell lysates were subjected to Western blot with anti-FLAG antibody. An asterisk indicates a non-specific signal of anti-FLAG antibody. cDNAs with a CTG or ATG, but not CGG, initiation codon were translated into 21-kDa protein (arrow). To make C-terminally FLAG-tagged cDNAs, an antisense primer composed of the sequences encoding the C-terminal 8 amino acids of mouse NPW followed by the FLAG epitope peptide, DYKDDDDK, was used. A point mutation at the putative initiator codon (CTG to ATG or CGG) was introduced by PCR using the sense primers with these mutations. pcDNA3.1(+) was used as an expression vector. CHO cells were transiently transfected with 30 μg of each plasmid DNA using Lipofectamine 2000 (Invitrogen). Cell extracts were resolved by SDS/PAGE and processed for Western blotting with the anti-FLAG antibody (Sigma), using the chemiluminescence detection kit (Pierce).1. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. (1997) Protein Eng. 10, 1–6.