FIGURE 1.

Analysis of the NGF and the BDNF pro-domain binding to the mammalian VPS10p receptors by SPOT analysis. A peptide library containing a total of 2181 peptides represented by 734 from sorLA, 403 from sorCS3, 389 from sorCS1, 382 from sorCS2, and 273 from sortilin as overlapping 16-mer peptides of the five human VPS10p-domain-containing receptors sortilin (CAA66904, 831 aa), sorLA (AAC50891, 2214 aa), sorCS1(AAM43811, 1179 aa), sorCS2 (Q96PQ0, 1159 aa), and sorCS3(Q9UPU3, 1222 aa). The membrane was incubated either in the presence of 10 μg/ml HisS-NGFpro (A), 10 μg/ml HisS-BDNFpro (B), or in the absence of ligand (C). Detection was carried out by incubation of the membrane with an HRP-conjugate of S-protein that recognizes the S-peptide tag of bound NT pro-domains. Each receptor library is cornered at the upper left and lower right corner by three control peptides equivalent to the S-peptide tag (amino acid sequence KETAAAKFERQHMS). The specific binding site for both HisS-NGFpro and HisS-BDNFpro (A and B), which is not seen for the control experiment in absence of ligand (C), is indicated for the three sortilin peptides (SPOT numbers 67–69; red box).