Fig 9.

The CD98-positive endocytic structures are positive for phosphoinositol-3-phosphate (PI3P) and Rab5. (A) Colocalization of vaccinia MV-containing CD98⁺ vesicles with PI3P. CD98^+/+ MEFs were incubated for 60 min at 4°C with FITC-conjugated anti-CD98 antibody (green) and WR-A4-mCherry (red) at an MOI of 20 PFU/cell; subsequently shifted to 37°C for 15 min; and washed with acidic buffer to remove the surface-bound antibody. The cells were fixed and stained for intracellular PI3P-containing macropinosomes (cyan) as described in Materials and Methods. Arrowheads point to the MV-containing CD98⁺ vesicles that are positive for PI3P. The large insets in each panel are enlarged views of the areas outlined by the small squares. (B) Colocalization of vaccinia MV-containing CD98⁺ vesicles with an endosome marker, Rab5. CD98^+/+ MEFs were treated with an anti-CD98 antibody (green) and WR-A4-mCherry (red) as described above for panel A. The cells were fixed, permeabilized, and stained for an endosome marker, Rab5 (cyan). Arrowheads point to the MV-containing CD98⁺ vesicles that are positive for Rab5.