CD146 is required for VEGF-mediated signal transduction. (A) Phosphorylation of VEGFR-2 induced by VEGF was determined after HUVECs were cotransfected with CD146-siRNA and pcDNA3.1-CD146. The quantification of the relative p-VEGFR-2/VEGFR-2 index is shown. At least 3 independent assays were performed. (B) AKT activation induced by VEGF was measured after HUVECs were cotransfected with CD146-siRNA and pcDNA3.1-CD146. (C) P38 activation. (D) NF-κB activation induced by VEGF after rescuing CD146 expression. Western blots were quantified by measuring the band density, which was then normalized to GAPDH. Bar graphs (means ± SEM) present normalized values from at least 3 independent experiments. (E) Immunofluorescence showing the role of CD146 in NF-κB nuclear translocation. NF-κB was detected using specific anti-p65 (red) and anti-p50 Abs (red, scale bar indicates 20 μm). *P < .05; **P < .01; ***P < .001. NS indicates not significant.