Effects of NgBR knockdown on VEGF-stimulated phosphorylation of Akt and Akt-dependent endothelial cell migration. (A-B) NgBR knockdown has no effect on AP-VEGF binding on the surface of HUVECs. After 48 hours of treatment with NS siRNA (siRNA-NS) or NgBR siRNA (siRNA-NgBR), HUVECs were incubated with 10nM AP or AP-VEGF at 4°C for 2 hours. AP was applied as controls to determine the nonspecific binding on the HUVEC surface. (A) The bound AP or AP-VEGF was detected using the Blue Substrate kit (Vector Laboratories). (B) The bound AP or AP-VEGF was extracted with Triton X-100, and AP activity was colorimetrically quantified using p-nitro-phenyl phosphate (Sigma-Aldrich) as substrate. (C) NgBR knockdown reduced the VEGF/AmNogo-B–stimulated phosphorylation of Akt and ERK in HUVECs. Quiescent siRNA-treated HUVECs was stimulated with 100 ng/mL VEGF/AmNogo-B for 30 minutes. The phosphorylation of Akt (S473) and ERK (p42/44) was determined by Western blot analysis as described in “Western blot analysis.” (D) Constitutively activated myrAkt restores the VEGF-induced migration of HUVECs treated with NgBR siRNA. Cell migration was examined in modified Boyden chambers in response to VEGF (100 ng/mL; n = 3). *P < .05, vs NS + VEGF; #P < .05, vs Ctrl + VEGF. (E) NgBR coding-region cDNA restores the VEGF-induced migration of HUVECs treated with NgBR siRNA. Cell migration was examined in modified Boyden chambers in response to VEGF (100 ng/mL; n = 3). *P < .05, vs NS + VEGF; #P < .05, vs Vector + VEGF + siNgBR. Vector, transfected with plasmid DNA vector containing the internal ribosome entry site (pIRES)–neo empty vector; NgBR, transfected with pIRES-neo vector carrying NgBR coding-region cDNA. siRNA, 10nM.