Nogo-B mediates ICAM-1–induced src and Pyk2 activation in MLECs, but does not affect VE-cadherin or src localization. (A) Nogo-A/B^−/− and WT MLECs were treated with TNF-α as above, followed by ICAM-1 cross-linking for 5 minutes, 10 minutes and 15 minutes. Lysates were analyzed by immunoblotting with antibodies and the phospho/total ratio determined for each pathway. (B) Immunofluorescence microscopy of VE-cadherin (green) and src (red) in TNF-α–stimulated HDMECs in the absence or presence of Nogo-B before (basal) and after ICAM-1 cross-linking (ICAM1-XL, 15 minutes). Blue reflects 4,6-diamidino-2-phenylindole, dihydrochloride staining of nuclei. Bar = 20 μm. The images in panel B were captured at a 0.3-μm slice thickness (z-stack) using a Zeiss Axiovert epifluorescence microscope and a 63× oil-immersion objective, followed by deconvolution using Openlab software. In panel C, the colocalization of src and VE-cadherin was quantified using the Pearson correlation. Data are representative of at least 3 experiments. XL, cross-linking.