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Fig. 3. F508 N894D/N900D cannot escape ER quality control. (A) Western blot of CFTR, CFTR N894D/N900D, F508 and F508 N894D/N900D expressed in BHK-21 cells. Lysates were separated by SDS-PAGE and CFTR was detected after transfer to nitrocellulose by mouse monoclonal anti-CFTR antibody 570. (B) F508 N894D/N900D does not interact with calnexin. F508 N894D/N900D was immunoprecipitated by incubation with anti-CFTR antibody 596 crosslinked to Dynabeads. CFTR and calnexin (CNX) were detected by western blotting using mouse monoclonal antibody 596 for CFTR or rabbit polyclonal antibody SPA860 for calnexin. Molecular weight marker positions (kDa) are indicated on the left. (C) Immunofluorescence microscopy of CFTR and F508 glycosylation variants in permeabilized cells. Immunostaining was performed on permeabilized BHK-21 cells using anti-CFTR mouse monoclonal antibody 570, followed by goat anti-mouse IgG Alexa Fluor 488 conjugate. Calnexin was stained to visualize the ER compartment using rabbit anti-calnexin antibodies followed by goat anti-rabbit IgG Alexa Fluor 568 conjugate. (D) Visualization of cell-surface CFTR on non-permeabilized cells by applying antibody HA11 to detect the external HA epitope in EL2 of CFTR variants. Cells were grown at 37°C or incubated at 27°C for 48 hours in the presence of 2 mM sodium butyrate to promote cell-surface expression of Extope-F508 CFTR. (E) cAMP-stimulated ³⁶Cl^– efflux measurements of stably expressing BHK-CFTR cells. Stimulation cocktail was added (+) at time 0. Each point represents the average of three independent samples and standard deviations are indicated. (F) Immunostaining of CFTR and F508 glycosylation variants in virally transduced well-differentiated primary human airway epithelial cells. All pools of intracellular CFTRs were stained on frozen sections of cultures grown at 37°C with HA11 antibody followed by goat anti-mouse IgG Alexa 488 conjugate. (G) Labeling of apical CFTR in virally transduced well-differentiated primary human airway epithelial cells. Cultures were incubated at 27°C for 48 hours and the apical pool of CFTR variants was labeled with HA11 antibody; cultures were then frozen in OCT and frozen sections labeled with goat anti-mouse Alexa 488 conjugate. Scale bars: 10 µm.

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