FIGURE 7:

Endocytosis of E-cadherin is specifically disrupted by loss of RalB but not RalA. (A, B) RalB knockdown results in decreased endocytosis of E-cadherin. Indicated cell lines were subjected to a Ca²⁺ switch after seeding on 24-mm Transwell filters and were biotinylated 9 h following HCM addition. Three filters of each cell type were not reduced (NR), reduced with no 37°C incubation (T-0), and incubated for 30 min at 37°C (T-30). Avidin pulldowns were completed, and E-cadherin immunoblots were performed with total and pulldown samples. Immunoblots were visualized with a ChemiDoc-It Imaging System and quantified with Visionworks software. (C) Transferrin internalization rate is not affected by Ral knockdown. ¹²⁵I-Transferrin was bound to cells infected with human transferrin receptor and warmed for indicated times (data points). Both internal and surface ¹²⁵I-transferrin were quantified and applied to a model in which clearance from the surface into early endosomes is represented by the equation . The lines represent best-fit curves generated by the model (shCtrl, solid line; shRalA, dashed line; shRalB, dotted line). (D) Inhibition of dynamin during TJ assembly causes a TER profile that phenocopies RalB knockdown. A Ca²⁺ switch was performed with MDCK II cells treated with DMSO or a dynamin inhibitor (dynasore, 80 μM). Triplicate TER readings of triplicate filters were recorded every hour for 20 h. (E) Dynasore inhibits endocytosis of transferrin. MDCK cells expressing human transferrin receptor were treated with DMSO or 80 μM dynasore. Samples were processed for immunofluorescence after addition of fluorescent human transferrin. Arrows indicate origin of the xy-slice within the z-series. Error bars, SD of triplicate samples. *p < 0.05. Bars, 20 μm.