Nogo-B deficiency impairs ICAM-1 cross-linking–induced VE-cadherin phosphorylation but not ICAM-1 clustering, stress fiber formation, and MLC phosphorylation. (A) Tyrosine phosphorylation of VE-cadherin after ICAM-1 cross-linking. HDMECs were transfected with Nogo-B siRNA or control siRNA were stimulated with TNF-α (10 ng/mL) for 24 hours, serum-starved, and incubated with ICAM-1 antibody 15.2 (5 μg/mL) for 30 minutes, followed by cross-linking for the indicated times. VE-cadherin immunoprecipitates were then analyzed by immunoblotting against phosphorylated tyrosine (4G10, PY20 clone). (B) The amount of tyrosine-phosphorylated VE-cadherin was quantified by densitometry from 4 independent experiments and is expressed as the fold increase of untreated controls. (C) Whole-cell lysates (WCLs) from HDMECs treated as described were immunoprecipitated for phosphotyrosine using the antibody PY20 or the 4G10 clone, and immunoprecipitates were analyzed by immunoblotting against VE-cadherin. The phosphorylation of VE-cadherin on ICAM-1 cross-linking was reduced in Nogo-B siRNA compared with control siRNA–treated HDMECs. (D) The activation of additional pathways (P-410 Pyk2, Pyk2, P-419-src, src, P-861-FAK, FAK, P-MAPK^42/44, MAPK, P-MLC, MLC) were examined after ICAM-1cross-linking. Densitometric quantification of phospho/total for each pathway is below. (E) The loss of Nogo-B did not impair ICAM-1 clustering or stress-fiber formation. HDMECs transfected with Nogo-B siRNA or control were stimulated with TNF-α (10 ng/mL) for 24 hours, and surface ICAM-1 (green) and F-actin (red) examined after ICAM-1 cross-linking for 1 hour. Bar = 20 μm. Images were captured using a Zeiss Axiovert epifluorescence microscope and a 63× oil-immersion objective. XL, cross-linking.