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Figure 5


Fig. 5. Effects of cholesterol depletion on an interaction between Flag-NPC1 and wild-type SKD1. Transfected COS cells were cultured in cholesterol-rich or cholesterol-depleted medium with or without LDL for 16 hours. (A) Co-precipitation of His6-SKD1wt with Flag-NPC1. Cells were transfected with His6-SKD1wt together with Flag-NPC1 constructs. Anti-Flag immunoprecipitation (IP) products were analyzed by immunoblotting (IB) with the indicated antibodies. Molecular weights are given on the left (kDa). (B) Cell fractionation. Cells were transfected with Flag-NPC1 and His6-SKD1wt constructs. Membrane fractions (100,000 g pellet) of cell homogenates were fractionated on an Opti-prep gradient. The fractions, together with the membrane fractions applied (input), were analyzed by immunoblotting with the indicated antibodies. Shown are representative results that were reproduced three times. (C) Intracellular localization of Flag-NPC1 and GFP-SKD1wt. Cells expressing Flag-NPC1 and GFP-SKD1wt were stained with anti-Flag antibody, and bound antibody was visualized with Alexa Fluor 546-conjugated secondary antibody. Results shown are the representative images obtained with a confocal microscope. Shown in the insets are the enlarged images of the indicated areas.





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