Fig. 4. Effects of blockade of endogenous Sema4d on metanephroic development. (A) Western blot analysis demonstrating the efficacy of a Sema4d function-blocking antibody (anti-Sema4d), but not preimmune serum (pre.; control), in attenuating Sema4d-induced RhoA activation in a GST-RBD-based assay in HEK293T cells transfected with plexin B1 (plxnB1) and PDZRhoGEF (PRG). Anti-Myc-positive bands in western blotting serve as loading controls. (B,C) Typical examples of the anti-calbindin-immunoreactive ureteric tree (green) in sister metanephroi from E12 embryos cultured in the presence of an anti-Sema4d antibody (C) or preimmune serum (B, control). (D,E) E12 metanephroi cultured with the anti-Sema4d antibody showed significantly more branching (D), but were the same size (E), when compared with metanephroi cultured with preimmune serum (control). ^*P<0.05, Student's paired t-test. Scale bars: 250 µm.