FIG. 2.

Internalization of ³⁵S-labeled rUEVs. The Caco-2 monolayers grown in 24-well plates (4 × 10⁵ cells/well) were washed three times with cold DMEM and chilled at 4°C. The plates were divided into two groups to measure the specific binding and internalization. The specific binding was measured as shown in Fig. 1 (left column). To measure the value of internalization, the other group of plates was incubated with 10 μg of ³⁵S-labeled rUEVs per well for 1 h at 4°C with gentle agitation. After washing three times with cold DMEM, the plates were transferred to 37°C for 1 h to allow the bound rUEVs to internalize into the cells. The cells were treated with proteinase K (500 μg/ml) for 30 min at 4°C and solubilized with RIPA buffer, and their radioactivity was measured. The internalization value was determined by subtracting the value of the remaining rUEVs on the cell surface after the proteinase K treatment (right column). Each assay was done in triplicate. Each column represents the mean value (with error bar).