FIGURE 2:

Transcytosis is impaired in cells expressing GTP-bound/Q77L or GDP-bound/N132I rab17. (A) Total cell lysates were prepared from WIF-B cells expressing FLAG-tagged WT, GTP-bound/Q77L, or GDP-bound/N132I rab17 and immunoblotted with anti-FLAG antibodies. The monosumoylated form of rab 17 is indicated (sumo rab17). Molecular weight markers are indicated on the left of each immunoblot in kDa. (B) Cells expressing wild-type, GTP-bound/Q77L, or GDP-bound/N132I rab17 were basolaterally labeled with antibodies specific for the extracellular epitopes of the indicated apical proteins at 4°C. Cells were additionally infected with recombinant adenoviruses expressing pIgA-R in panels g–i. After excess antibodies were washed away, antibody–antigen complexes were chased for 90 min (a–c) or 60 min (d–i) at 37°C. Cells were fixed, permeabilized, and labeled with secondary antibodies to detect the transcytosed proteins. Arrows indicate subapically accumulated transcytosing proteins in cells expressing mutant rab17. Images are representative of at least three experiments. Bar = 10 μm.