Plexin B1 is the endothelial receptor required for Sema4D-angiogenic activity. (A) mRNAs obtained from untreated and Sema4D-treated HUVECs were retrotranscribed and amplified with Plexin B1 or CD72-specific primers (as described in “Materials and methods”). cDNA obtained from lymphocytes of a patient with chronic lymphocytic leukemia was used as positive control for CD72; MDA-MB-435 cells expressing Plexin B1 were used as positive control for Plexin B1. As shown, Plexin B1 (top blot) and CD72 (middle blot) are expressed in HUVECs, and Sema4D stimulation did not significantly up-regulate their expression. (B) Migration assay of wild-type HUVECs and HUVECs transfected with Plexin B1–specific siRNA. As shown, an impairment of cell migration in response to Sema4D was observed in wild-type cells in the presence of inhibitory α-Plexin B1 antibodies and in cells displaying decreased expression of Plexin B1 because of siRNA expression. The negative controls varied by experimental condition: in the experiments with inhibitory α-Plexin B1 antibodies, as a control, wild-type (WT) cells were treated with flow-through; in the experiments with Plexin B1–specific siRNAs, control cells were transfected with GFP-specific siRNA. Neither the antibody treatment nor the expression of siRNA affected HUVEC response to VEGF-A₁₆₅. Data are shown as mean ± SD. (C) Sprouting assay performed in the same conditions described in panel B. A reduction of Sema4D-induced sprouting was observed in the presence of inhibitory α-Plexin B1 antibodies and in cells expressing Plexin B1 siRNAs. In addition, in this assay, response to VEGF-A₁₆₅ was not affected. (D) Quantification of the sprouting assay shown in panel C. Data are shown as mean ± SD. (E) Western blot analysis of HUVECs transfected with Plexin B1–specific siRNAs. The reduction of Plexin B1 has been quantified at approximately 70%. HSP70 indicates heat shock protein 70. (F) Binding assay. HUVECs transfected with siRNAs specific for Plexin B1 displayed a strong impairment in Sema4D-AP binding ability (iv), compared with wild-type HUVECs (ii). The AP enzyme alone was used as negative control (i,iii). Images were captured with a 10×/0.40 air objective lens.