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Figure 6


Fig. 6. Effects of cholesterol depletion on ubiquitylation of endogenous NPC1 and its interaction with SKD1 in human skin fibroblasts. Cells from a control subject or patients with NPC1 or NPC2 disease were cultured in cholesterol-rich or cholesterol-depleted medium for 16 hours. (A) Expression of NPC1 and SKD1. 0.5% CHAPS extracts were analyzed by immunoblotting (IB) with antibodies against the indicated proteins. (B) NPC1 ubiquitylation and its association with SKD1. 0.5% CHAPS extracts were subjected to anti-ubiquitin (P4D1) immunoprecipitation (IP) and the immunoprecipitated products were analyzed by immunoblotting with the indicated antibodies. Molecular weights are given on the left (kDa). (C) Cell fractionation. Membrane fractions (100,000 g pellet) of cell homogenates were fractionated on an Opti-prep gradient. All results shown are representative and were reproduced at least twice.





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