Fig. 1: Localisation of NET1ΔC on the cell surface and different patterns of NET1ΔC-HSPG binding vs NET1ΔC-GAG mediated interactions.
a Transmission immune electron micrographs of ultrathin sections of HEK-293 cells overexpressing NET1ΔC. A control image (left) is compared to addition of exogenous HO-dp10 (middle) and HEK-293 cells overexpressing of heparanase (right). The top and bottom row compare low- and high-resolution images. Black dots represent immune-gold labelled anti-NET1 mAB molecules depicting NET1ΔC deposits. The scale bars represent 200 nm (upper) and 20 nm (lower). b Statistical analysis of the appearance of immuno-gold labelled anti-NET1 mAb on the HEK-293 cell surface were analysed by counting the gold particles/μm2 on cellular profiles (N = 30). The results of an ordinary one-way ANOVA Tukey multiple comparisons test are indicated: ****, P < 0.0001. Mean and standard deviation (SD) are indicated. c ELISA style binding studies show protein-protein interactions of the heparan sulphate proteoglycans (HSPGs) glypican-3/5, SMOC-1/2, and testican-1/2 to immobilised NET1ΔC protein. NET1ΔC was coated and incubated with serial dilutions of strep-tagged HSPGs (0.5–500 nM). Binding was detected using a strep-HRP antibody. Error bars indicate the standard deviation from the mean of N = 3 technical repeats. d Negatively stained transmission electron micrographs of NET1ΔC-glypican-3 interactions. Glypican-3 exhibits a small globular central core protein to which three extended GAG chains are attached. Variable numbers of NET1ΔC molecules are bound to different locations along the GAG chains. The scale bar represents 20 nm. e ELISA style binding studies of immobilised glypican-3 to soluble NET1ΔC alone and together with ectodomains of DCC, neogenin and UNC5B, demonstrate NET1ΔC-mediated dependence receptor complex formation. NET1ΔC and receptor ectodomains were used in serial dilution from 0.5 to 500 nM (1:1 ratio of ligand to receptor). Binding was detected with a polyclonal, lab-made NET1 antibody. Error bars indicate the standard deviation from the mean of N = 3 technical repeats. f Competitive assays underline that a single-chain GAG competes with NET1ΔC-glypican-3 complex binding. Here, NET1ΔC (500 nM) was pre-incubated with HO-dp20 (1–1000 nM) and binding was detected against immobilised glypican-3 using NET1 antibody. Error bars indicate the standard deviation from the mean of N = 3 technical repeats.
