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Fig. 6. The P4 prominin-1-containing membrane particles are distinct from exosomes carrying the tetraspanin CD63. (a) 24-hour-conditioned medium of Caco-2 cells grown for 10 days post-confluency was subjected to differential centrifugation as in Fig. 2a, and the resulting pellets were analysed by immunoblotting for prominin-1. (b) 24-hours-conditioned medium of Caco-2 cells grown for 14 days post-confluency was centrifuged for 30 minutes at 10,000 g, and the resulting supernatant, containing the P4 particles, was concentrated and subjected to equilibrium sucrose gradient centrifugation. Aliquots of the fractions (1.5%) and the pellet (50%) were analysed by immunoblotting for prominin-1. (c,d) Negative staining and prominin-1 immunogold (12 nm) labelling EM of the P4 particles present in fraction 7 of the sucrose gradient. Note the presence of both prominin-1-positive (arrows, indicated only in c) and prominin-1-negative (open arrows) small vesicles. (e,f) Negative staining EM of prominin-1 (6-nm gold) and CD63 (12-nm gold) double-immunogold labelled particles present in fraction 7 of the sucrose gradient. Note that the small vesicles showing prominin-1 immunoreactivity are distinct from those showing CD63 immunoreactivity.
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