Effects of MCD treatment on primary T cells. (A) Purified splenic T cells (> 98% CD3+) were treated with the indicated concentrations of MCD, and their adhesion to immobilized ICAM-1 was analyzed as in Figure 1A. Open bars represent no PMA stimulation, solid bars represent PMA-stimulated (50 ng/mL) cells, and gray bar represents PMA stimulation with anti–LFA-1 blocking antibody (TIB213). Cells treated with 10 mM MCD and 2 mM MnCl2 (10 + Mn) were included. BSA instead of ICAM-1 was used as a control. The results are representative of 4 independent experiments, each done in triplicate. Error bars indicate SD. (B) Purified splenic T cells treated (+) or not (-) with 10 mM MCD were stained for the indicated cell-surface molecules and analyzed by flow cytometer as in Figure 1B. (C) Splenic T cells were treated with (+) or without (-) 10 mM MCD, lysed with 1% or 0.05% Triton X-100, subjected to sucrose gradient centrifugation, and indicated molecules in each fraction were detected by Western blotting as in Figure 1D.
