Figure 3. CBLN family binding and cell-based DCC interaction affinity measurements.

A) Sensograms are shown from SPR analysis of immobilized DCC-Fc (left; 15,082 response units) and negative control Robo3-Fc (right; 9,833 response units) with CBLN1, CBLN2, CBLN3 and CBLN4 injected as analytes at 10 µg/ml. Robust binding was detected to CBLN4 (black trace), while no binding signal was detected for the other Cerebellin family members (gray traces). B) A radio-lignad assay was used to determine the affinity of CBLN4-Fc (left) and gD-Netrin-1 (right) to DCC transiently transfected into HEK293T cells. ¹²⁵I-Labeled ligand was allowed to bind transfected cells in the presence of increasing amounts of unlabeled ligand. Calculated K_D values are indicated on the graphs and are representative of 3 independent experiments.