Characterization of Tmtsp protein and specific antibody. (A-B) Specificity of anti–mouse Tmtsp mAb. To verify the specificity of the anti-Tmtsp mAb, cell surface proteins of BW5147 cells infected with a Tmtsp retrovirus (BW5147/Tmtsp) and mock control cells (BW5147/control) were biotinylated, and the total cell lysates were used for immunoprecipitation with anti-Tmtsp mAb, followed by detection with streptavidin-HRP. Tmtsp protein and the putative proteolytic products are indicated by arrowheads (A). The specificity of the mAb was also confirmed by flow cytometric analysis (B). Open curve indicates anti-Tmtsp; gray shaded curve, isotype control. (C) Binding assay with recombinant netrin-1 protein. COS7 cells transiently transfected with DCC and FLAG-Tmtsp were incubated with chick netrin-1 (VI · V)–Fc. Receptor expression (green) and netrin-1-Fc (red) were merged. Scale bar: 50 μm.