Figure 2
Effect of the C-terminal truncation on agonistic mAb Zt/g4-induced RON or RON160 phosphorylation and dimerization. (A) 3T3-RON or other cells (4 × 106 cells/sample) were stimulated with or without mAb Zt/g4 (2 nM) in serum-free conditions for 10 min. Half of the cells were biotinylated for cell surface proteins. Labeled RON, RON160, or their variants were immunoprecipitated with mAb Zt/c1 and detected in Western blotting using avidin-conjugated antibodies (top panel). The other half of the cells were lysed and immunoprecipitated by Zt/c1. Phosphorylated proteins were detected by mAb PY-100 (middle panel). To ensure equal amounts of samples used, cell lysates were directly analyzed for actin in Western blotting (bottom panel). (B) Cells were stimulated as above and then treated with a permeable cross-linker followed by cell surface biotinylation [13]. The proteins were immunoprecipitated and detected in Western blot analysis as described in (A). Data shown here are from one of three experiments with similar results.