Fig. 5.

PECAM-1 is enriched in caveolar fractions. A: plasma membrane from mouse PMVEC was isolated from post nuclear homogenate using a Percoll gradient shows PECAM-1 expression in the caveolin-1-enriched fractions (fractions 5–8). All lanes were immunoblotted with membrane marker flotillin. B: caveolae obtained from plasma membranes of mouse PMVEC after Percoll gradient centrifugation and sonication. Intact caveolae vesicles were separated from other membrane domains (noncaveolar) by immunoaffinity methodology using magnetic beads. Noncaveolar fraction shows a caveolin-1 signal as larger vesicles with caveolin-1 are often not pulled down by this immune-assay and remain unbound to the magnetic beads. Proteins from each fraction were separated by SDS-PAGE, transferred to nitrocellulose membranes and immunoblotted using anti-PECAM and caveolin-1 antibodies.