Fig. 3

sv6D as a mimetic of GalNAc. a Inhibition by sv6D of binding of multivalent GalNAc-PAA (GlycoTech Corp., Frederick, MD) to recombinant rat CLEC10A and human ASGPR-1. The reaction mixture included approximately 200 pmoles of biotinylated GalNc-PAA and increasing concentrations of peptide. The figure includes values ± S.D. from 3 independent experiments. b Biotinylated sv6D or GalNAc-PAA were incubated with rabbit antiserum raised against the 6D sequence (NQHTPR) conjugated to KLH. Binding was detected with streptavidin-peroxidase conjugate. Similar data were obtained in 2 experiments. c Inhibition by EGTA of binding of sv6D (red, yellow) or svL4 (dark green, light green) to CLEC10A and ASGPR-1, respectively. EGTA (1 mM) was added to the final concentrations indicated to assays. Retention of bound CLEC10A was determined by incubation with biotinylated anti-CLEC10A (goat IgG, R&D Systems) and detection with streptavidin-peroxidase conjugate (blue). Similar data were obtained in 3 experiments. d Binding of svL4 and sv6D to human recombinant CLEC10A and ASGPR-1 as a function of concentration of peptide in the assay. The figure is representative of 4 separate assays. K_D values ± S.D. from reciprocal plots of these data are provided in the text