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Figure 6


Fig. 6. Analysis of exosomes isolated from K562 cav cell culture media. (A) 20 µg cell debris obtained by centrifugation of culture medium from biotinylated-K562 cav cells was subjected to a 40%-5% continuous sucrose gradient in the absence of detergent, then analyzed by streptavidin and TfR2 immunoblotting. Positions of molecular size markers in kDa are indicated on the right. (B) 20 µg exosomes obtained as described in Materials and Methods were biotinylated then layered on a 30% to 10% continuous sucrose gradient and analyzed by streptavidin and TfR2 immunoblotting. In both cases equal volumes of fractions 1-11 were loaded. Sucrose densities were obtained for each fraction by refractometry. (C) Left panel, 3 µg of plasma membrane (PM) and 3 µg of LDTI prepared from biotinylated K562 cav cells or 1 µg of biotinylated exosomes (EXO) from K562 cav cells were analyzed by streptavidin immunoblotting. Note the differential pattern of biotinylated proteins displayed as exosomes, LDTI and PM. Right panel, equal protein amounts of plasma membrane (PM), LDTI and exosomes (EXO) prepared from K562 cav cells were analyzed by western blotting for CD45 and TfR1 expression.





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