Nogo-B cofractionates, interacts with VE-cadherin, and translocates toward the junction on ICAM-1 cross-linking. (A) ICAM-1, VE-cadherin, and Nogo-B sedimentation in raft membrane fractions analyzed by sucrose gradient fractionation. Nogo-B siRNA–treated or control treated HDMECs were stimulated with TNF-α (10 ng/mL for 24 hours) and then fractionated. β-Actin was used as a marker for non-raft fractions and Cav-1 for rafts and caveolae. (B) TNF-α–treated HDMECs were lysed and p120 or Nogo-B coimmunocoprecipitated. Samples were then analyzed by Western blotting using anti–VE-cadherin, anti-p120, and anti–Nogo-B antibodies, respectively. Isotype-matched, nonimmune antibody served as the control for immunoprecipitation experiments. The immunoblot shown is representative of 3 independent experiments. (C) Immunofluorescence labeling of Nogo-B (green) and F-actin–rich stress fibers (phalloidin, red) before and after ICAM-1 cross-linking for 30 and 60 minutes. Cells were stimulated with TNF-α (10 ng/mL) before ICAM-1 cross-linking. Nuclei were stained with 4,6-diamidino-2-phenylindole, dihydrochloride (blue). Bar = 20 μm. (D) Immunofluorescence colabeling of Nogo-B (green) and VE-cadherin (red) before (i-iii) and after ICAM-1 cross-linking (iv-vi). Nuclei were stained with 4,6-diamidino-2-phenylindole, dihydrochloride (blue). Bar = 20 μm. Images were captured with a Zeiss Axiovert epifluorescence microscope and a 63× oil-immersion objective. XL, cross-linking.