FIGURE 3.

CAR location in lipid microdomains at the plasma membrane and during internalization. A, CAR was found in flotillin-1⁺ fractions, a lipid microdomain protein in mouse cortical neuron extract subjected to biochemical isolation through OptiPrep gradient. Cortical neurons were incubated with 10 mm methyl-β-cyclodextrin (MβCD), a known lipid microdomain destabilizator, and subjected to lipid microdomain isolation. Methyl-β-cyclodextrin triggered flotillin-1 and CAR relocalization in lower fractions. Western blots are representatives of 3 independent experiments. B, CAR did not exit lipid microdomains after ligand binding and internalization. Lipid microdomain isolation was performed on cortical neurons incubated either with FK^CAV on ice, or shifted to 37 °C for 20 min after incubation on ice with FK^CAV, 10,000 pp/c CAV-2, or 10,000 pp/c of HAdV5. Western blots are representatives of three independent experiments. C, filipin blocked CAR internalization. Hippocampal neurons were treated with 5 μg/ml of filipin for 30 min prior to FK^CAV/Tf or FK^CAV/CTB incubation on ice for 20 min. Cells were washed and then shifted to 37 °C for 1 h to trigger internalization. In contrast to Tf, CTB and FK^CAV did not enter after filipin treatment. D, quantification of three independent experiments showed that filipin efficiently blocked FK^CAV internalization (n = 3, ≥45 cells counted for each condition; results are expressed as mean ± S.E. ***, p value <0.001 versus control). Scale bar, 5 μm.