(Downloading may take up to 30 seconds.
If the slide opens in your browser, select File -> Save As to save it.)
Click on image to view larger version.


Figure 3


Fig. 3. Misassembled rather than dysfunctional CFTR mutants cause cholesterol redistribution. (A) Influence of reduced temperature on maturation of different CFTR variants. Western blots showing maturation of CFTR and CFTR variants {Delta}F508, 1410X, D572N and G551D grown at 37°C and 27°C. Maturation of CFTR mutants {Delta}F508 and 1410X CFTR is rescued by low temperature. D572N CFTR is retained at the ER at high or low temperature and the severe-disease-causing mutation G551D, which prevents CFTR channel activation, is processed normally at 37°C and 27°C. Cell lysates were separated by 6% SDS-PAGE, transferred to nitrocellulose and proteins were detected by immunoblotting using anti-CFTR antibody 596. (B) Free cholesterol (Chol.) is elevated in CFTR-processing mutants. Free cholesterol content of cells expressing CFTR variants at 27°C was quantified after cellular lipid extraction from equal numbers of cells and thin-layer chromatographic analysis to compare amounts of cholesterol in cells expressing mutant CFTR with the amount of cholesterol detected in cells expressing wild-type CFTR. Values are representative of three independent experiments. Cells expressing {Delta}F508 and 1410X CFTR show a significant increase in cholesterol content (*P<0.05) in comparison with cells expressing wild-type CFTR. (C) Cholesterol distribution in these variants grown at 27°C. Microscopy showing filipin staining of CFTR and CFTR variants {Delta}F508, 1410X, D572N and G551D grown at 27°C. The percentage of cells showing perinuclear Golgi staining is shown in a histogram. At least 75 cells were counted and values are representative of three independent experiments. (D) CFTR inhibitor I-172 is without effect on cholesterol distribution. Filipin staining of BHK-21 cells expressing CFTR untreated (control) or treated with 10 µM CFTR inhibitor I-172 for 1, 8 or 24 hours. Neither the percentage of cells showing Golgi staining by filipin nor the relative filipin intensity changes in the presence of the inhibitor. The histogram shows the percentage of cells with perinuclear Golgi staining. At least 75 cells were counted and values are representative of three independent experiments.