<p>(<b>A</b>) PNGase F treatment of Semaphorin-7A. Biotinylated Semaphorin-7A was incubated with PNGase F for 10, 30 or 60 minutes. Enzyme-treated and untreated Semaphorin-7A were resolved by SDS-PAGE under reducing conditions and detected by Western blotting using Streptavidin-HRP. (<b>B</b>) Binding of MTRAP to PNGase F-treated Semaphorin-7A was indistinguishable from untreated Semaphorin-7A using the AVEXIS assay. MTRAP was used as the prey against Semaphorin-7A baits. (+) = positive control, (−) = negative control. Bar graphs show mean ± SEM, n = 3. (<b>C</b>) PNGase F treatment did not quantitatively influence MTRAP binding to Semaphorin-7A using SPR. Three concentrations of purified monomeric MTRAP were injected over flow cells immobilised with PNGase F-treated and untreated Semaphorin-7A. Dissociation rate constants (<i>k</i><sub>d</sub>) were calculated to be 0.063±0.00007 s<sup>−1</sup> for PNGase F treated, and 0.061±0.00006 s<sup>−1</sup> for untreated Semaphorin-7A, by fitting a first order dissociation model to the washout phase of the binding curves. Shown are the normalized, averaged values ± SEM, n = 3. (<b>D</b>) MTRAP does not interact with sulphated glycoconjugates. Purified monomeric Semaphorin-7A, chondroitin sulphate A, chondroitin sulphate C, dextran sulphate, heparin and heparan sulphate were injected at 1 mg/ml over MTRAP immobilised on a streptavidin-coated sensor chip.</p
