Figure 5.

Effect of agents that dissipate endosomal pH gradients on MAL-FLAG internalization. (a–c) COS-7 cells were transiently transfected with MAL-FLAG. After 48 h, cells were incubated with anti-FLAG mAb for 1 h at 4°C and then for 30 min at 37°C in the presence of 10 μM monensin (a), 100 μM chloroquine (b), or 50 mM NH₄Cl (c). (d–f) Cells were pretreated with monensin (d), chloroquine (e), or NH₄Cl (f) for 30 min before binding of the anti-FLAG antibody, and the drugs were maintained during both the incubation with the primary antibody and the 30-min incubation at 37°C. Finally, cells were fixed, permeabilized, and incubated with the secondary antibody. (g) Cells were treated with 100 μM chloroquine for 2.5 h at 37°C, fixed, permeabilized, and subjected to immunofluorescence analysis with mAb 9E10. Note that panel g shows the distribution of the total MAL-FLAG protein, whereas panels a–f show only the distribution of the MAL-FLAG originally present on the cell surface. Bar, 10 μm.