Fig. 4. Co-localization of PrP^C and PECAM-1 in raft/caveolae-like microdomains in brain endothelial cells. (A) RBE4 cells were harvested by scraping and submitted to subcellular fractionation in the presence of sodium carbonate as described in the Materials and Methods. Fractions (1-10) were collected from the top of the gradient and proteins in each fraction were concentrated by precipitation with 10% trichloroacetic acid. Equal volumes (15 µl) of concentrated fractions were analyzed by SDS-PAGE and processed for immunoblot analyses. Membranes were incubated with antibodies to caveolin-1 (0.5 µg/ml), PrP^C (SAF32, 1 µg/ml), PECAM-1 (M20, 1 µg/ml) or ß-catenin (1 µg/ml). PrP^C and PECAM-1 are detected in fraction 4 containing raft/caveolae-like microdomains; PrP^C appears as a typical smear, as seen in the whole cell lysate (panel B). Other forms of PrP^C were detected in fraction 10, which contains the majority of cellular proteins, including ß-catenin; these forms of PrP^C probably correspond to intermediate glycosylation forms. (B) Whole RBE4 cell lysate was treated (+) or not (–) with PNGase F and processed for western blot analyses using SAF32 anti-PrP^C antibodies (1 µg/ml). Following PNGase F treatment (+) a single band of approximately 25 kDa is detected, corresponding to unglycosylated PrP^C. The results are representative of three independent experiments.