Figure 2
Tunicamycin prevents glycosylation of OSTα but does not affect its plasma membrane localization or its transport function. Tunicamycin (1 μg/ml) was added to HepG2 cells 5 hrs after the addition of 50 μM CDCA and incubation continued for a total of 15 hrs. Cells were then extracted with RIPA buffer for PAGE and Western blotting, fixed for immunofluorescence, or subjected to the transport assay as described in Methods. (A) Western blot analysis of cell lysates indicates that the molecular weight of the OSTα subunit was reduced from ~36 kD to ~28–30 kD (see arrows), indicating that this subunit is glycosylated. The molecular weight of the OSTβ subunit was not significantly changed and β-actin was used as a loading control. (B) Immunofluorescence for OSTα shows that tunicamycin treatment did not prevent expression of the alpha subunit on the plasma membrane. (C) Transporter function as assessed by 3H-estrone 3-sulfate uptake was also not affected by treatment with tunicamycin. n = 3