Figure 3.

Sema4D-dependent inhibition of R-Ras activity and ECM-mediated cell migration involves the endogenous Plexin-B1 receptor. (A) PC12 cells were pretreated with 5 μg/ml of a mouse monoclonal antibody against the extracellular ligand binding region of Plexin-B1 (PlexB1 Ab) or the control mouse IgG2b (Control Ab). PC12 cells were seeded onto noncoated or collagen-coated (10 μg/ml) dishes with or without Sema4D in the plating media, and relative R-Ras activity was determined as described in the legend to Fig. 2 A. (B) PC12 cells were pretreated with 5 μg/ml of the monoclonal antibody against Plexin-B1 and were subsequently subjected to the transwell assay in either the presence or absence of Sema4D. Migrated cells were visualized by the staining of crystal violet. (C) Relative cell migration was determined by the number of the migrated cells normalized to the total number of cells. Results are the means ± SEM of three independent experiments. Bar, 1 mm.