Fig. 2. Anti-ICAM-1 immunobead uptake is inhibited by dominant-negative dynamin constructs. EAhy926 cells were transfected with 1.5 µg of DNA encoding either wild-type (A-C) or dominant-negative (K44A; e, PH*; f) dynamin-2. Nontransfected cells are shown in (D). Twelve hours post-transfection, cells were stimulated with TNF- for 36 hours and then incubated for 2 hours at 37°C with anti-ICAM-1 immunobeads. The cells were then washed, fixed and surface-bound particles were counterstained with TxR goat anti-mouse IgG. The cells were then permeabilized and stained with rabbit anti-6-His antibody followed by Alexa Fluor 350 goat anti-rabbit IgG to identify transfected cells expressing dynamin (A). The corresponding phase-contrast image is shown in (B). Merged images corresponding to representative samples of transfected (C,E,F) or control (D) cells are shown, where single-labeled, internalized immunobeads are green (arrows) and double-labeled immunobeads on the cell surface are yellow (arrowheads). Blue fluorescence of transfected cells is omitted in panels (C,E,F) to enable better visualization of red and green fluorescence. (G) The percentage of immunobead uptake was calculated as described as mean±s.d. *P<0.05.

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