Fig. 4. Misprocessed ABC protein F728 MRP1 also affects cellular cholesterol distribution. (A) F728 MRP1 is a temperature-sensitive misfolding mutant. F728 MRP1 matures when cells are grown at low temperature. Western blot of lysates from BHK-21 cells expressing MRP1 or F728 MRP1. BHK-21 cells stably expressing MRP1 or 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) F728 MRP1 escapes ER quality control at low temperature and proceeds to the cell surface. Immunofluorescence microscopy showing localization of MRP1 and 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 F728 MRP1 show redistribution of cholesterol similar to cells expressing F508 CFTR. BHK-21 cells expressing MRP1 or 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.