Fig. 4. Anti-ICAM-1 conjugates are not internalized by clathrin or caveolae-mediated endocytosis. TNF--activated HUVEC were untreated (A-C), potassium-depleted (D-F), or treated for 30 minutes at 37^oC with 1 µg/ml filipin (G-I), or 3 mM amiloride (J-L). The cells were incubated in the presence or absence of inhibitors for 1 hour at 37°C with fluorescent transferrin (Tf: A,D,G,J), fluorescent cholera toxin (CT: B,E,H,K) or anti-ICAM-1 immunobeads (C,F,I,L), then fixed and counterstained to double-label surface-bound material (yellow, arrowheads). As shown, potassium depletion specifically inhibited transferrin uptake by clathrin-mediated endocytosis (D), filipin specifically inhibited caveolar uptake of cholera toxin (H) and amiloride specifically inhibited uptake of anti-ICAM-1 immunobeads (L).