Disruption of lipid rafts and inhibition of LFA-1 activation by MCD treatment. (A) EL4 cells (left panel) or T28 cells (right panel) were incubated with (▪) or without (□) 50 ng/mL PMA in the presence of the indicated concentrations of MCD in serum-free HBSS and incubated at 37°C for 30 minutes, and their adhesion to immobilized soluble ICAM-1 was analyzed. Cells treated with or without PMA, 10 mM or 20 mM MCD, and 2 mM MnCl₂ were also tested (shown as 10 + Mn and 20 + Mn). PMA-activated cells blocked with anti–LFA-1 were tested as specificity control (▦). For control cell adhesion, BSA was immobilized in place of ICAM-1. The results are representative of 5 independent experiments, each done in triplicate. Error bars indicate SD. (B) Flow cytometric analysis of control (-) and 10 mM MCD-treated (+) EL4 cells. Ganglioside GM1 was stained with FITC-conjugated CTxB. All other molecules were stained with the appropriate mAb with secondary FITC-conjugated antibodies. Ctrl shows unstained control. (C) EL4 cells were solubilized with indicated concentrations of Triton X-100 and subjected to sucrose gradient centrifugation. Proteins in the sucrose gradient fractions were separated by SDS-PAGE, and CD18 was detected by Western blotting. Fractions 2 and 3 are low-density fractions and contain lipid rafts, whereas fractions 5 to 8 are high-density fractions, and P indicates pellet. (D) EL4 cells were either treated (+) or not (-) with 10 mM MCD as in panel A, solubilized with 0.05% Triton X-100, subjected to sucrose gradient centrifugation, and analyzed by Western blotting for the indicated molecules. The numbers indicate sucrose gradient fractions. Fractions 2 and 3 are low-density fractions containing lipid rafts. CD18, Thy1, and CD45 were detected by specific mAb and horseradish peroxidase–conjugated secondary anti–rat Ig antibody. GM1 was detected by horseradish peroxidase–conjugated CTxB.