GLUT1 and cholesterol distribution along sucrose-gradient fractionation of M07e cells treated or not with MBCD.

<p>(<b>A</b>) M07e cells treated (or not) with 10 mM MBCD for 20 min were lysed with 1% Triton X-100 at 4°C and separated by sucrose density-gradient ultracentrifugation as described in the Materials and Methods section. Equal volume aliquots of each fraction were subjected to SDS-PAGE and Western blotting. Flotillin-2 and Lyn were used as markers for DRM fractions; CD71 for non-DRM fractions. (<b>B</b>) Typical profile of protein concentrations in gradient fractions after ultracentrifugation. Protein content was determined as described in the Materials and Methods section. (<b>C</b>) M07e cells were pre-incubated at 37°C for 16 hours with [³H]-cholesterol (0.1 µCi/mL) in cell culture medium. Cells exposed (or not) to 10 mM MBCD for 20 min were lysed with 1% Triton X-100 at 4°C and subjected to sucrose density-gradient ultracentrifugation as previously described. [³H]-cholesterol content of each fraction collected was quantified by liquid scintillation counting. Results are expressed as means ± SD of three independent experiments, each performed in triplicate. ***<i>P</i><0.001, significantly different from untreated cells.