Fig. 4.

Caveolin-3, APP, and the presenilins are colocalized within caveolae membranes in cultured cells.A, CRT astrocytoma cell line.Left, CRT cells were subjected to detergent-free subcellular fractionation that separates caveolae from the bulk of cellular and cytosolic proteins, as described by Song et al. (1996s).Fraction 5 represents the caveolae-rich fraction, andfractions 8–12 contain ∼99% of the total cellular protein. Note that APP (top, middle) and caveolin-3 (bottom) were highly enriched within the caveolae fraction. APP was detected by immunoblotting using AC-1 and 4G8 antibodies. Right, CRT cells were subjected to a second independent detergent-free approach used to purify caveolae membranes (the Optiprep method). Each lane contains 2 μg of protein. APP, PS-1, PS-2, and caveolin-3 were all detected primarily in the caveolae membrane fraction (lane 4), whereas they were barely detectable in the noncaveolar fraction of the plasma membrane (lane 3). B, COS-7 cells. Cells cotransfected with APP and caveolin-3 were subjected to subcellular fractionation as described in A(left). Note that APP and caveolin-3 were confined primarily to the caveolae membrane fraction (seefraction 5).