Fig. 8.

Lack of affinity of netrin-G1 to netrin receptors. UNC5H3/RCM, with its cytoplasmic domain replaced with ECFP, was transiently expressed in COS7 cells. The receptor protein expression was detected with ECFP fluorescence (blue;A, C, E). DCC was expressed in HEK293EBNA cells and detected using mouse monoclonal anti-DCC antibody and Alexa 488-conjugated anti-mouse IgG (green; G,I). In the same field, binding of myc-tagged chick netrin-1 (Netrin-1/Myc) on the cells was detected by immunocytochemistry using a monoclonal anti-myc antibody (9E10) and Alexa 546-conjugated anti-mouse IgG (B) in the case of UNC5H3/RCM, and rabbit polyclonal anti-myc antibody and Alexa 546-conjugated anti-rabbit IgG (H) in the case of DCC (red). However, secreted forms of myc-tagged netrin-G1a (sNetrin-G1a/Myc) and Netrin-G1d (sNetrin-G1d/Myc) did not show binding to the cells expressing both receptors, even at the higher concentrations (D, F, J). Similar results were obtained with cells expressing UNC5H1 or UNC5H2 receptors. sNetrin-G1c and sNetrin-G1e also did not show binding to cells expressing any type of receptor examined (data not shown).K represents relative concentrations of test ligands used in binding experiments, revealed by anti-myc immunoblotting. Shown are representative results from one of three independent experiments. Scale bar, 10 μm.