Figure 3. RhoA and Akt are necessary for Plexin-B1-mediated activation of NF-κB and promotion of endothelial cell migration and tube formation.

A. Immunoblot for phospho-I-κB (top panel) and total I-κB (middle panel) in cells expressing chimeric receptors coding for the extracellular portion of Trk-A fused to the wild-type intracellular segment of Plexin-B1 (wtPB1), Plexin-B1 lacking the PDZ binding motif (ΔPDZ) or Plexin-B1 mutated in the RasGAP domain (RasGAP mut), treated with NGF with and without the Rho inhibitor C3 toxin (C3) and the PI3K inhibitor LY294002 (LY), for the times indicated. GAPDH was used as a loading control (lower panel). B. NF-κB reporter assay performed on control treated HUVEC or HUVEC treated with Sema4D, with or without C3 toxin or LY294002 (LY), with fluorescence expressed as arbitrary units (AU). Error bars represent the standard deviation from three experiments. C. Migration assays on HUVEC towards BSA or Sema4D in the presence of control media, or media containing C3 or LY294002 (LY). Quantification of migration is shown in the bar graph in the lower panel. Error bars represent the standard deviation from six wells (*, p<0.05). D. HUVEC were plated on reconstituted basement membrane material in serum free media and treated with vehicle control (control) or Sema4D, with or without co-treatment with C3 toxin (C3) or LY294002 compound (LY), and examined for formation of capillary tubes. Representative photographs are shown. Quantification of the results observed in the tubulogenesis assay are shown in the right panel (error bars represent the standard deviation from three independent experiments; *, p<0.05).