Figure S3.

mUnc5A and mUnc5C binding results, protein co-expression analysis, related to Figure 3

(A) SPR results show binding of hGPC3^core protein to mouse Unc5A and C ectodomain. The apparent K_D (K_Dcalc) for the wild type protein interaction was calculated using a 1:1 binding model and is indicative only. Bmax, R² and the units of ligand immobilised on the flowcell surface are indicated. The N241Q mutant protein (hGPC3^coreUG) does not show binding.

(B) We used a cell-based assay to show that hGPC3^core, but not the mutant, binds to mUnc5C expressed on cells.

(C) SPR results show no binding of hGPC3^core protein to Unc5(A)–(D)^GU mutant proteins. Corresponding binding curves using wild type Unc5 proteins are shown in Figure S1D.

(D) Western blot analysis using anti-HA to visualise HA-tagged Unc5 constructs expressed in cell aggregation assays.

(E) Same samples as in panel D, but here visualising Flag-GPC3.

(F) Same as panel D, but using anti-actin control.

(G) Cell-surface staining using anti-HA and anti-Flag was performed to complement the total protein expression analysis shown in panels (D)–(F), and to include additional conditions. Representative images are shown. Scale bars = 30 μm.

(H) Quantification of the experiments shown in panel F.