Figure 10.

Internalization of transiently expressed wild-type TGN38, S331T, and S331E in the absence or presence of PI3K inhibitor. Cos-7 cells were transiently transfected with wild-type TGN38, S331T, or S331E. Monoclonal antibody to TGN38 (2F7.1) was added to the tissue culture medium, and antibody uptake was allowed to occur for 2 h as described in the legend to Figure 8. During the final 30 min of uptake, cells were incubated in the absence (A, C, and E) or presence (B, D, and F) of the PI3K inhibitor LY294002 to identify late endosomes. Cells were fixed and processed for immunofluorescence microscopy as described in the legend to Figure 8. Regardless of the construct expressed, internalized 2F7.1 appeared in numerous peripheral vesicular structures (arrows). Only in cells expressing S331E did internalized 2F7.1 accumulate in swollen vesicular structures in response to LY294002 treatment (F, arrows). Asterisks, TGN. Bars, 10 μm.