Role of Plasma Membrane Caveolae/Lipid Rafts in VEGF-Induced Redox Signaling in Human Leukemia Cells
Figure 2
Isolation and identification of membrane caveolae/lipid rafts by detergent extraction and sucrose gradient centrifugation in B1647 cell line. (a) Cells were lysed with 1% Triton X-100 at 4°C and separated by sucrose density-gradient ultracentrifugation as described in Section 2. Equal aliquots of each fraction were subjected to SDS-PAGE and Western blotting. Flotillin-2, caveolin-1, and Lyn were used as markers for caveolae/lipid raft fractions and CD71 for nonlipid raft fractions. (b) Typical profile of protein concentrations in gradient fractions after ultracentrifugation. Protein content was determined as described in Section 2. (c) Cells were preincubated at 37°C for 16 hours with [3H]-cholesterol (0.1 μCi/mL) in cell culture medium then exposed (or not) to 10 mM CD for 20 min, 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. °: significantly different from each other; *: significantly different from untreated cells; **: significantly different from untreated cells; ***: significantly different from untreated cells.
