Figure 4.

Nanobodies enhance or disrupt GPC3-Unc5 interaction

(A) SPR binding data using hGPC3^core and Nano^glue.

(B) As in (A), with Nano^break.

(C) The equilibrium values from experiments shown in panels A and B, and equivalent values from using mGPC3⁴⁸⁸ (Figures S4E and S4F). K_D values were calculated assuming 1:1 binding.

(D) Unc5B pull downs with immobilized hGPC3^core using Nano^glue and Nano^break.

(E and F) Equilibrium SPR data (raw data in Figures S4E and S4F) confirms that Nano^glue enhances, and Nano^break weakens, GPC3-Unc5D binding.

(G) Cell-cell aggregation assay using soluble Nano^break and Nano^glue. Representative images.

(H) Quantification of cell-cell aggregation experiments.

(I) E15.5 dissociated cortical neurons were grown on alternate stripes (red and black) containing Fc, GPC3^core, or GPC3^coreUG.

(J) Quantification beta-III-tubulin+ (green) pixels on red stripes: Fc (=Fc/Fc control), GPC3 (=hGPC3^core/hGPC3^coreUG). We performed equivalent stripe assays also for HeLa, N2A and SY5Y cells, using DAPI for quantification. ^∗∗∗∗p < 0.0001, Student T-tests.

(K) Results from stripe assays in the presence of streptavidin (Ctrl) or streptavidin-nanobody complexes (Nano^glue or Nano^break).

We performed one-way ANOVA with Tukey’s post hoc tests (H) and (K). NS, not significant; ^∗p < 0.05; ^∗∗∗p < 0.001; ^∗∗∗∗p < 0.0001. Scale bars represent 100 μm (G) and 90μm (I).

See also Figure S4.