Fig. 6.

HM-ICAM-1 displays impaired interactions with the actin cytoskeleton. HUVECs were treated with TNF-α (10 ng/ml, 6 h) in the presence of absence of α-mannosidase inhibitors swainsonine or kifunensine. A: ICAM-1 was immunoprecipitated as described in experimental procedures for interaction with the Ezrin-Radizin-Moesin (ERM) complex. Whole cell lysates (input) and immunoprecipitated proteins were analyzed by Western blot analysis. B: cells were treated as before and some cells underwent antibody-mediated ICAM-1 clustering. ICAM-1 was immunoprecipitated as described in materials and methods for interaction with caveolin-1. Whole cell lysates (input) and immunoprecipitated proteins were analyzed by Western blot analysis. C: cells were treated and some cells underwent antibody-mediated ICAM-1 clustering (X-ICAM). Lysates were separated into Triton X-100 soluble (S) and insoluble (I) fraction and analyzed by Western blot. Data are representative of at least 3 different experiments. D: cells were treated and underwent antibody-mediated clustering of ICAM-1. After fixation, cells were stained with fluorescently tagged secondary antibody and surface expressed patterns of ICAM-1 were assessed. Data are representative of at least 3 different experiments.