FIG 1.

Infected GNPTAB^−/− HeLa cells lack active lysosomal hydrolases. (A) The active form of cathepsin D is absent from infected GNPTAB^−/− cells. Immunoblot of procathepsin and mature forms of cathepsin D in cells at 3 dpi. (B and C) CCVs in GNPTAB^−/− cells are deficient in cathepsin D. Cells were infected for 3 days with mCherry-NMII, fixed with PFA, and fluorescently stained for cathepsin D using BODIPY FL pepstatin A and the CCV membrane using anti-LAMP1 antibody. Images were analyzed for intensity of BODIPY FL pepstatin A per CCV. (D and E) CCVs in GNPTAB^−/− cells lack sulfatase activity. Cells were infected for 3 days with mCherry-NMII, incubated for 18 h with SulfGreen, and imaged live. Images were analyzed for intensity of SulfGreen per CCV. Graphs represent the means ± standard deviations (SD) of ≥150 cells from 3 independent experiments. Statistical significance was determined by Student's t test (****, P < 0.0001). Nuclei were stained with Hoechst stain. Scale bar, 10 μm.