Figure 2
Zt/f2 binds specifically to MRS in the RON β-chain extracellular sequence. A) Zt/f2 does not bind RONsema. Recombinant RONsema (0.5 μg per sample) from transfected cells (Ma et al., 2010) was immunoprecipitated with 2 μg/ml Zt/f2 or other mAbs with Protein G-Sepharose. Samples were analyzed under non-reduced conditions by Western blot analysis using antibodies to RON extracellular sequences (Wang et al., 1994). B) Schematic representation of RON and its variants. General structures of RON are illustrated on the left. The α- and β-chains are indicated. Deleted regions in individual variants are marked with arrows. TM, transmembrane segment; MRS, maturation-related sequences; and TK, tyrosine kinase domain. C) Zt/f2 does not interact with RON165 and RON155. Cellular proteins (300 μg per sample) from NIH3T3 cells expressing RON, RON160, RON165, RON155, or RON110 were immunoprecipitated with 2 μg/ml of Zt/f2. Samples were detected by Western blot analysis using rabbit IgG antibody to RON C-terminal peptide (Xu et al., 2004). D) Zt/f2 does not bind to RON165 and RON155 in intact cells. NIH3T3 cells expressing individual RON variants (0.5 × 106 cells per sample) were permeabilized as previously described (Lu et al., 2007) and then incubated with 2 μg/ml of Zt/f2. Fluorescence was detected by FITC-coupled goat anti-mouse IgG. E) MRS peptide blocks Zt/f2 binding to RON. Zt/f2 (2 μg/ml) was first mixed with or without 1 μM of synthetic peptide for 30 min and then incubated with 3T3-RON cells. Cell surface fluorescence was determined as described above.