Fig. 5. Apical targeting by the deglycosylated Na⁺/K⁺-ATPase ß-subunit was mediated via the indirect transcytosis pathway. (A) Polarized Hep G2 cells were treated with monoclonal anti-Na⁺/K⁺-ATPase ß-subunit antibody at 4°C for 15 minutes. After washout of the primary antibody, the cells were warmed to 37°C for the time periods indicated, then fixed and stained with fluorescently labelled secondary antibody. Within the 60 minute observation period, no fluorescent signal appeared at the bile canaliculi (arrows). (B) The same experiments were applied to the Hep G2 cells treated with tunicamycin. The fluorescent signals were originally found only along the basolateral membrane (arrowheads); over time, there was a progressive increase in fluorescence at the apical canalicular domain (double arrowheads). (C) The percentage of bile canaliculi (BC) that contained the fluorescence generated from the mistargeted Na⁺/K⁺-ATPase ß-subunit was calculated in the control () and tunicamycin-treated cells (). The bile canaliculi were recognized by F-actin staining; those also positively stained for Na⁺/K⁺-ATPase ß-subunit were identified. At least 500 bile canaliculi obtained from approximately 20 microscopic images were included in the calculation. Results shown are the means ± s.d. of three independent experiments. Bars, 10 µm.