Role of Plasma Membrane Caveolae/Lipid Rafts in VEGF-Induced Redox Signaling in Human Leukemia Cells
Figure 3
Effect of cholesterol depletion from plasma membrane on VEGFR-2 localization, ROS generation, and glucose transport activity in B1647 cells. (a) Cells in the presence or absence of human serum (HS) and/or pretreated with 5 mM CD for 20 min 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, Western blotting, and revealed for anti-VEGFR-2 (210 kDa). A representative blot is shown. (b) Cells in the presence or absence of human serum (HS) and/or pretreated with 5 mM CD for 20 min were incubated with 5 μM DCFH-DA and ROS intracellular level was measured spectrofluorimetrically as described in Section 2. (c) Cells in the presence or absence of human serum (HS) and/or pretreated with 5 mM CD for 20 min were incubated with DOG mixture and glucose uptake was assayed by liquid scintillation counting as described in Section 2. (d) Cells in the presence or absence of human serum (HS) and incubated (or not) in PBS at 37°C with 5 mM CD for 20 min were fixed in 3% (w/v) paraformaldehyde for 15 min. Cells were then immunolabelled with anti-Glut1 (N-20) antibody (raised against an extracellular domain of Glut1, therefore, evidencing that Glut1 is present on the cell surface), treated with fluorescent FITC-conjugated secondary antibody, and visualized using immunofluorescence microscopy.
