Figure 1. Heparin or deletion of domain C prevents nonspecific adsorption of netrin-1 but preserves DCC binding. A, Domain structure of netrin-1. Domain V and VI bind to netrin-1's receptor DCC, while domain C contributes to substrate adsorption through its many positively charged amino acids. B–D, HEK293 cells were transfected with GFP-tagged DCC (DCC-GFP) and incubated with either 1 μg/ml full-length mCherry-tagged netrin-1 (B), 1 μg/ml full-length NTN1-mCherry and 2 μg/ml heparin (C), or 1 μg/ml NTN1dC (D) for 90 min. As highlighted by the cells that do not express DCC (white arrows), the presence heparin or deletion of domain C restricts binding to only the cells that express DCC. E–G, Differential interference contrast (DIC) images (bottom) and unmodified fluorescent intensity images (top) of spinal commissural neurons incubated for 15 min with, either 1 μg/ml NTN1-mCherry (E), 1 μg/ml NTN1-mCherry with 2 μg/ml heparin (F), or 1 μg/ml NTNdC-mCherry (G). Relative to full-length netrin applied alone, there is a decrease in background binding in the presence of heparin (37%, p < 0.01, n = 40) or when domain C is deleted (20%, p < 0.01, n = 40) and there is distinct labeling of both axon and growth cone. H, I, ELISA detecting the myc tag within recombinant, full-length netrin-1 when media containing 200 ng/ml are incubated with either a collagen cushion for 16 h (H) or a polylysine-coated tissue culture well (I) for time periods of 15 min or less. Significant binding of full-length netrin is seen to collagen gels (n = 16, p < 0.01) and to polylysine dishes within 5 min (n > 15, p < 0.05) compared with media alone. Inclusion of 2 μg/ml heparin significantly reduced the association of netrin-1 to collagen by 79% (n = 16, p < 0.01) and to a polylysine-coated dish by 90% after 15 min. Fluorescent intensity comparison based on average intensities of 10 μm2 areas from images of equal exposure (5 s). Scale bars: B, 50 μm; E, 10 μm.