Fig. 5

A, on day 0, HEK293 cells were seeded in 35-mm dishes. On day 1, the cells were transfected with β-galactosidase, Ostα, Ostβ, or Ostα-Ostβ. On day 2, the cells were washed and incubated in Hanks’ buffered saline containing 25 μm [³H]taurocholate for 30 min at 37 °C. Cells were then washed and lysed to determine cell-associated radioactivity and protein. The uptake is expressed as pmol of taurocholate transported/mg of protein (mean ± S.D., n = 3) and is corrected for the background uptake in β-galactosidase transfected cells (15.6 ± 0.6). Taurocholate uptake was significantly increased following co-transfection with Ostα plus Ostβ (p < 0.001). B, on day 0, HEK293 cells were seeded in 60-mm dishes. On day 1, the cells were transfected with Ostα, Ostβ, or Ostα-Ostβ. On day 2, the cells were lysed, and 100 μg of cell protein was incubated in the absence (lanes 1, 2, 7, 8) or presence of Endo H (lanes 3, 4, 9, 10), or PNGase F (lanes 5, 6, 11, 12). Samples were then subjected to immunoblotting analysis for Ostα (lanes 1–6) and Ostβ (lanes 7–12). The migration of the precursor (p), mature glycosylated (m), and unglycosylated (u) forms are indicated. C, on day 0, HEK293 cells were seeded onto glass coverslips. On day 1, the cells were transfected with Ostα (panels 1 and 4), Ostβ (panels 2 and 5), or Ostα-Ostβ (panels 3 and 6). After 24 h, the cells were fixed with 3.7% formaldehyde/PBS and permeabilized using a solution of 1% bovine serum albumin and 0.1% saponin in PBS. The cells were then stained with mouse M2 anti-FLAG to detect Ostα (red), rabbit polyclonal anti-Ostβ (green), and To-Pro-3 (blue) to visualize nuclei and then viewed using laser scanning confocal microscopy.