Figure 2

Released claudin proteins are present in exosome particles. A, BG-1 culture media was centrifuged sequentially and analyzed by immunoblotting for the expression of claudin-4. Claudin-4 was sedimented by a 100,000 g centrifugation (100 K Centrif.) but not by 10,000 g centrifugation (10 K Centrif.). B, Sucrose gradient analysis of the 100,000 g pellet. Following separation of the pellet on a sucrose gradient, ten successive fractions of increasing densities were collected and analyzed by immunoblotting for the expression of the indicated claudin proteins. The density of the various fractions was measured and is indicated on top of the lanes. C, Transmission electron microscopy image of exosomes released from ovarian cancer cells. Exosomes isolated from BG-1 culture media by the 100,000 g centrifugation were fixed, negatively stained, and observed by TEM. The exosomes observed are 40–100 nm in diameter and exhibit the typical cup-shaped morphology. D, Indirect immunogold labeling with anti-claudin-4 antibody followed by a secondary antibody coupled to 6-nm gold particles. The gold particles (dark dots on the figure) clearly localize to the exosomes.