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. 2017 Jun 16;113(6):1342–1352. doi: 10.1016/j.bpj.2017.05.032

Figure 1.

Figure 1

CD63 and CD9 are released on distinct EVs. (A) Confocal z-stacks in orthogonal view of HEK293 cells stably expressing mGFP-CD9 on the plasma membrane and mCherry-CD63 on intracellular vesicles. Scale bars, 10 μm. (B) Flow chart of EV isolation and sucrose density gradient flotation protocols. (C) For the indicated proteins, 8 μg of total cellular protein and 1 μg of 100k EVs prepared from HT-29 cells were separated by SDS-PAGE and immunoblotted. 100k EVs from HEK293 cells showed similar protein profile (not shown). (D) Distribution of tetraspanin proteins after flotation of 100k EVs from HEK293 cells into linear sucrose density gradients. The x axis indicates the density of each fraction as determined by refractometry. Fractions were immunoblotted for CD63 (black line) and CD9 (gray line) and quantitated, and the values were plotted as the percent of total signal across all fractions. The experiment shown is representative of four biological replicates. To see this figure in color, go online.