American Society of Hematology

Figure 5.

$Figure 5. Detection of GM1 ganglioside as a marker for lipid rafts. (A) GM1 on intact cells. Cells were treated with FITC-ChB in a direct binding assay and the cells were analyzed on FACScan. Namalwa/MDR1 cells (1) in media, (2) stained with FITC-ChB, (3) pretreated with anti-CD19, or (4) pretreated with anti–P-gp prior to staining with FITC-ChB. (B) Distribution of GM1 ganglioside in the lipid fractions isolated by sucrose gradient ultracentrifugation of the Triton X-100 lysates of Namalwa/MDR1 cells. Cells were incubated for one hour at 37° C in the absence or presence of HD37, UIC2, or RFB4 (15 μg/106 cells), and 10 μM verapamil or 10 mM MBCD, lysed, and fractionated in a sucrose gradient. Combined raft fractions and soluble fractions from 5 × 108 cells were resolved (25 μL/lane) by 12% SDS-PAGE under nonreducing conditions and immunoblotted with ChB–horseradish peroxidase.$

Detection of GM1 ganglioside as a marker for lipid rafts. (A) GM1 on intact cells. Cells were treated with FITC-ChB in a direct binding assay and the cells were analyzed on FACScan. Namalwa/MDR1 cells (1) in media, (2) stained with FITC-ChB, (3) pretreated with anti-CD19, or (4) pretreated with anti–P-gp prior to staining with FITC-ChB. (B) Distribution of GM1 ganglioside in the lipid fractions isolated by sucrose gradient ultracentrifugation of the Triton X-100 lysates of Namalwa/MDR1 cells. Cells were incubated for one hour at 37° C in the absence or presence of HD37, UIC2, or RFB4 (15 μg/10⁶ cells), and 10 μM verapamil or 10 mM MBCD, lysed, and fractionated in a sucrose gradient. Combined raft fractions and soluble fractions from 5 × 10⁸ cells were resolved (25 μL/lane) by 12% SDS-PAGE under nonreducing conditions and immunoblotted with ChB–horseradish peroxidase.

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