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Figure 4


Fig. 4. Misprocessed ABC protein {Delta}F728 MRP1 also affects cellular cholesterol distribution. (A) {Delta}F728 MRP1 is a temperature-sensitive misfolding mutant. {Delta}F728 MRP1 matures when cells are grown at low temperature. Western blot of lysates from BHK-21 cells expressing MRP1 or {Delta}F728 MRP1. BHK-21 cells stably expressing MRP1 or {Delta}F728 MRP1 were grown either at 37°C or shifted to 27°C. Lysates were separated by SDS-PAGE, transferred to nitrocellulose and detected using mAb 897.2. (B) {Delta}F728 MRP1 escapes ER quality control at low temperature and proceeds to the cell surface. Immunofluorescence microscopy showing localization of MRP1 and {Delta}F728 MRP1 at 37°C and 27°C. Immunostaining was performed on cells permeabilized with 0.1% saponin using mouse mAb 897.2 followed by Alexa Fluor 488 goat anti-mouse IgG. (C) Cells expressing {Delta}F728 MRP1 show redistribution of cholesterol similar to cells expressing {Delta}F508 CFTR. BHK-21 cells expressing MRP1 or {Delta}F728 MRP1 were grown at 37°C or 27°C and stained with filipin. The percentage of cells showing Golgi staining is shown in a histogram. At least 75 cells were counted and values are representative of three independent experiments.





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