Fig. 2. Unglycosylated CFTR does not bind to calnexin but functions as a chloride channel. (A) Western blot of CFTR and unglycosylated CFTR created either by mutation of the glycosylation sites (N894D/N900D) or by expressing CFTR in tunicamycin-treated cells (+ Tunicamycin). CFTR and CFTR N894D/N900D variants were stably (left panel) or transiently (right panel) expressed in BHK-21 cells. Tunicamycin (5 µg/ml) was added directly after transient transfection to achieve complete inhibition of N-linked glycosylation. CFTR was detected in the microsomal membrane vesicle fraction by western blotting using mouse monoclonal anti-CFTR antibody 596. (B) CFTR N894D/N900D does not interact with calnexin. CFTR was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads (as indicated on the left). CFTR and calnexin (CNX) were detected by western blotting using mouse monoclonal antibody 596 for CFTR or rabbit polyclonal antibody SPA860 (Stressgen) for calnexin (as indicated on the right). Molecular weight marker positions (kDa) are indicated on the left. (C) The channel properties of unglycosylated CFTR are similar to wild-type CFTR. Single-channel measurements were performed using membrane vesicles prepared from stably transfected BHK-CFTR, BHK-N894D/N900D cells or from cells transiently transfected with CFTR that were treated with tunicamycin. Tunicamycin (5 µg/ml) was added directly after transient transfection to achieve complete inhibition of N-linked glycosylation.