Figure 5.
Overall protein composition of metabolically labeled exosomes and D1 cells. (A) Exosomes purified from the supernatant of metabolically labeled D1 cells were run on a 12% SDS gel (exos), together with lysate (including both cytosolic and membrane components) obtained from the whole cells (cells), cytosol (cyto), or total membranes (mb) prepared from the same cells in the absence of detergent. The dried gel was autoradiographed for 24 h (proteins >45 kD) or 48 h (proteins <45 kD). A pic profile of each lane was obtained with the NIH Image software. Stars point to proteins abundant in the cells and absent in exosomes, dots to proteins enriched in exosomes. (B) Metabolically labeled exosomes were subjected to flotation on a continuous sucrose gradient. The fractions were collected (density 1.08–1.28 g/ml), run on a 10% SDS-PAGE, and compared with exosomes that had not been subjected to flotation (Ex). All the proteins present in exosomes comigrate at the expected density (1.15 g/ml) (see Fig. 2 B; Raposo et al. 1996).