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Fig. 1. F508 CFTR perturbs intracellular cholesterol trafficking. (A) Cholesterol redistribution in cells expressing F508 CFTR is enhanced at reduced temperature. BHK-21 and CHO-K1 cells expressing CFTR and F508 CFTR were grown at 37°C and 27°C and stained with filipin. Bar, 10 µm. The histogram indicates the percentage of cells showing perinuclear Golgi-like staining versus punctate late endosomal/lysosomal staining. For quantification, at least 90 cells were counted from three different experiments and standard deviations are indicated. (B) F508 CFTR escapes ER quality control at reduced temperature. Western blot of lysates from BHK-21 cells expressing CFTR and F508 CFTR grown at 37°C and 27°C shows that F508 CFTR matures when cells are grown at low temperature. Cell lysates were separated by 6% SDS-PAGE and proteins were detected by immunoblotting after transfer to nitrocellulose using anti-CFTR antibody 596. Immunofluorescence microscopy on nonpermeabilized cells using mouse mAb HA11 to detect an external HA epitope in an expanded second extracytoplasmic loop of CFTR, followed by Alexa Fluor 488 goat anti-mouse IgG, confirms cell-surface localization of F508 CFTR in cells that were incubated at 27°C. BHK-21 clones stably expressing CFTR and F508 CFTR with HA epitope have been described earlier (Gentzsch et al., 2004).

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