Figure S4.

Nano^glue and Nano^break in SPR experiments and stripe assays, related to Figure 4

(A–D) Binding curves from SPR experiments. Unc5A-D receptor ectodomains were immobilised. Human GPC3^core or murine GPC3^ecto was injected using a 2-fold dilution series (top concentrations are 4.5 μM), in the presence of FC control protein, Nano^glue or Nano^break. The concentration of nanobodies was kept constant at 9 μM (with hGPC3^core), or 4.5 μM (with mGPC3^ecto). The concentration of FC control protein was kept constant at equivalent mg/ml concentrations.

(E and F) An analogous experiment was performed using immobilised nanobodies, and different concentrations of human GPC3^core or murine GPC3⁴⁸⁸ and Unc5D^ecto. Taken together, the results demonstrate that Nano^break competes with Unc5 for GPC3-binding, whilst Nano^glue strengthens the interaction. Calculated K_Ds for nanobody-GPC3 interactions are shown in Figure 4C. Given the unusual stoichiometry of the Unc5-GPC3 complex, we have not calculated K_D values from experiments containing also Unc5.

(G) Purified proteins were immobilised in a stripe pattern to assess their effect on the migration of cortical neurons. GPC3^core and GPC3^coreUG trigger strong cell repulsion, compared to neutral control protein (Fc).

(H) Quantification of the experiments shown in panel F. One-way ANOVA with Tukey’s post hoc tests.^∗∗∗p < 0.001.

(I) We performed GPC3^core/GPC3^coreUG stripe assays, but in the presence of streptavidin (CN) or streptavidin-nanobody complexes. Nano^break reduced the ability of neurons to distinguish between hGCP3^core and hGCP3^coreUG.

(J) Quantification of data shown in panel H. ^∗∗∗p < 0.001, two-tailed Student’s T test. Scale bar represents 90 μm (G) and (I).