FIGURE 1:

Basolateral-to-apical transcytosis is not altered by overexpression of wild-type rab17. (A) The characteristic, polarized hepatic morphology of WIF-B cells is shown in a phase image. (B) Total cell lysates were prepared from uninfected (control) WIF-B cells or cells expressing FLAG-tagged WT 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. (C) Uninfected cells or cells expressing wild-type rab 17 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 e and f (C). After excess antibodies were washed away, antibody–antigen complexes were chased for 0, 90, or 60 min as indicated at 37°C. Cells were fixed, permeabilized, and labeled with secondary antibodies to detect the transcytosed proteins. Asterisks mark the unlabeled bile canaliculi. Images are representative of at least three experiments. Bar = 10 μm. (D) Control (uninfected) WIF-B cells or cells expressing wild-type rab17 were basolaterally labeled for the indicated apical proteins and chased as described in C. Random fields were visualized by indirect immunofluorescence. From micrographs, the average pixel intensity of each marker at selected regions of interest placed at the apical or basolateral membrane of the same WIF-B cell was measured. The averaged background pixel intensity was subtracted from each value and the ratio of apical- (ap) to-basolateral (bl) fluorescence intensity was determined. Wild-type values were normalized to control values that were set to 100%. Values are expressed as the mean ± SEM. Measurements were performed on at least three independent experiments.