Effect of RTX on BCR translocation and on raft-associated cholesterol content. (A) DOHH2 cells were preincubated with F(ab′)2 RTX (10 μg/mL, 16 hours) and then stimulated with anti-IgG antibody (10 μg/mL, 5 minutes). Results are representative of 3 independent experiments. Vertical lines have been inserted to indicate a repositioned gel lane. (B) Cholesterol staining by filipin in RTX- or MβCD-treated and fixed RL cells (black histograms) compared with untreated and fixed cells (gray histograms). Results are representative of 3 independent experiments. (C) DOHH2 cells were treated with RTX (10 μg/mL, 16 hours), and cholesterol from raft and nonraft fractions was measured as described in “Methods.” Histograms are the mean of 3 independent experiments ± SD. *P < .005 compared with untreated cells. (D) RL cells were preincubated with MβCD (5mM, 30 minutes), and then BCR was stimulated with anti-IgM antibody (10 μg/mL, 5 minutes). Histograms represent Syk phosphorylation (Y525) expressed in MFI and are the mean of 3 independent experiments ± SD. *P < .005 compared with BCR-stimulated cells.