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Fig. 4. EDEM binds CFTR and accelerates its degradation, but neither the inhibition of EDEM interaction nor of calnexin interaction can rescue 
F508 CFTR. (A) CFTR was immunoprecipitated from cells transiently expressing HA-tagged EDEM by anti-CFTR monoclonal antibody 596 crosslinked to Dynabeads. EDEM was detected by western blotting using monoclonal antibody HA11 and CFTR was detected by antibody 596. (B) CFTR was expressed transiently in BHK-21 cells alone or together with EDEM using pcDNA3 CFTR and pCMV-SPORT2 EDEM, respectively. In the case of CFTR expression alone, similar amounts of empty control vector were co-transfected with the CFTR plasmid. (C) Immunofluorescence microscopy of F508 CFTR expressed in BHK-21 cells that were treated with various glycosidase inhibitors or tunicamycin. CAS (castanospermine, 0.2 mM) inhibits glucosidase I; DMM (1-deoxymannojirimycin, 0.5 mM) and kifunensine (0.2 mM) are inhibitors of mannosidase I; and tunicamycin (10 µg/ml) completely blocks N-glycosylation. The activity of these compounds was confirmed (supplementary material Fig. S3; Fig. 2A). Inhibitors were added to the growth media for 18 hours at the concentrations indicated and F508 CFTR was visualized on permeabilized cells using anti-CFTR monoclonal antibody 596 followed by goat anti mouse IgG Alexa Fluor 488 conjugate. Scale bar: 10 µm. (D) Cell-surface ELISA showing that glycosidase inhibitors do not rescue F508. BHK-21 cells expressing an externally tagged F508 CFTR (Gentzsch et al., 2004) were seeded in a 96-well plate at 40,000 cells per well. Twenty-four hours after seeding, cells were treated for 20 hours with CAS (0.4 mM), DMM (0.5 mM), KIF (kifunensine, 0.2 mM) or tunicamycin (10 µg/ml). Corrector compounds C3 (VRT-325) and C4 (corr-4a), which have been reported to partially rescue F508 CFTR (Loo et al., 2005; Pedemonte et al., 2005; Van Goor et al., 2006), were used as positive control at 20 µM each. Cells were fixed, labeled with HA11 antibody that recognizes the external tag, followed by goat anti-mouse IgG conjugated to X-Sight 761 and scanned with an infrared imaging system. A significant increase in the cell-surface pool of F508 CFTR was observed on treatment with C3 and C4 (*P<0.0001). Neither treatment with glycosidase inhibitors nor complete inhibition of N-glycosylation by tunicamycin had an effect that was significantly different from the no-drug control. Data represent the average of eight wells; bars indicate s.e.m. Statistical significance was determined using an unpaired Student's t-test. The blue line indicates the baseline level of the no-drug control.
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