FIGURE 6:

Transcytosing proteins accumulate in syntaxin 2–positive SAC structures in cells expressing sumo-deficient rab17. (A) Total cell lysates were prepared from WIF-B cells expressing FLAG-tagged WT or sumo-deficient/K68R rab17 and immunoblotted with anti-FLAG antibodies. The mono-sumoylated form of rab17 is indicated with an arrow. Molecular-weight markers are indicated on the left of each immunoblot in kDa. (B) Uninfected WIF-B cells were pretreated with 5 μM anacardic acid (AA) for 60 min at 37°C before APN antibody labeling at 4°C for 20 min. APN-antibody complexes were chased for 60 min in the continued presence of the drug. The amount of impaired transcytosis is indicated below the panel as the percent of control. Values are expressed as the mean ± SEM from at least three independent experiments. (C) WIF-B cells expressing sumo-deficient/K68R rab17 were basolaterally labeled with antibodies against 5′NT, and antigen-antibody complexes were chased for 60 min. Cells were fixed and double labeled for steady-state syntaxin 2 distributions. A merged image is shown in panel c. Arrows indicate subapically accumulated transcytosing proteins in cells expressing mutant rab17. Bar = 10 μm. The Mander’s coefficient of colocalization is indicated below the merged image. Values are expressed as the mean ± SEM from at least three independent experiments. (D) WIF-B cells expressing sumo-deficient/K68R rab17 were basolaterally labeled for 5′NT and ASGP-R (a) or APN (b) or APN and endolyn-78 (c) and allowed to continuously chase for 60 min. Cells were fixed and stained for the corresponding trafficked antibody–antigen complexes. In D (panel b), cells were labeled for steady-state distributions of EEA1. Merged images are shown. Arrows indicate subapically accumulated transcytosing proteins in cells expressing mutant rab17. Mander’s coefficients of colocalization are indicated below. Values are expressed as the mean ± SEM from at least three independent experiments. Bar = 10 µm. (E) Uninfected WIF-B cells (con) or cells expressing WT of K68R rab16 were lysed in ice-cold GTP binding buffer. Cleared lysates were mixed with GTP-agarose for 2 h at 4°C. Bound fractions were recovered, washed, and immunoblotted for rab17 using anti-FLAG epitope antibodies. The monosumoylated form of rab17 is indicated with an arrow. Molecular-weight markers are indicated on the left of each immunoblot in kDa. A representative immunoblot from three independent experiments is shown.