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Two volumes of washing buffer (25 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 M urea, 0.5 mM EDTA, 1 mM DTT, 1% (v/v) Tween-20 and 0.4 U l 1 Ribolock RNase inhibitor (Thermo Fisher Scientific)) were then added, and the pellet was washed by pipetting up and down 30 times and centrifuged at 4 C and 8,000 g for 1 min

[Note: This structure is called CURL, the compartment for uncoupling of receptor and ligand.] [5] The receptors can be recycled, whereas the lipoprotein remnants in the vesicle are transferred to lysosomes and degraded by lysosomal acid hydrolases, releasing free cholesterol, amino acids, fatty acids, and phospholipids