Fig. 1. Effects of cholesterol depletion on ubiquitylation of Flag-NPC1 expressed in COS cells. (A) Cellular cholesterol levels. Cells were cultured in cholesterol-rich medium (DMEM + 10% BCS) or cholesterol-depleted medium (DMEM + 10% LPDS supplemented with compactin and mevalonate) with or without LDL (20 µg/ml) up to 24 hours. Concentrations of total cholesterol in cell lysates were determined as described in the Materials and Methods. Each point represents the mean of duplicated determinations obtained in a single experiment. (B) Conjugation of myc/His₆-ubiquitin (myc/His₆-Ubi) to Flag-NPC1. Cells were transfected with expression constructs for myc/His₆-ubiquitin together with Flag-NPC1 or an empty vector. 48 hours after transfection, they were further cultured in the indicated medium. 0.5% CHAPS extracts were subjected to anti-Flag immunoprecipitation (IP) followed by immunoblotting (IB) with indicated antibodies. Molecular weights are given on the left (kDa). The arrowhead indicates the top of the separating gel and the asterisk indicates the heavy chain. (C) Co-purification of Flag-NPC1 with myc/His₆-ubiquitin. After incubation in the indicated medium for 6 hours, cell extracts were subjected to affinity purification with metal resin followed by immunoblotting with indicated antibodies. (D) Conjugation of endogenous ubiquitin to Flag-NPC1. Cells were transfected with Flag-NPC1 or an empty vector, cultured in the indicated medium for 6 hours and anti-Flag IP products were analyzed by indicated antibodies. (E) Effects of exogenous cholesterol on ubiquitylation of Flag-NPC1. Cells were transfected with myc/His₆-ubiquitin and Flag-NPC1, and cultured for 6 hours in cholesterol-rich or cholesterol-depleted medium supplemented with increasing concentrations of cholesterol. (F) Effects of U18666A. The transfected cells were cultured in the indicated medium for 12 hours in the presence or absence of U18666A. (G) Solubility of Flag-NPC1 to 1% Triton X-100. The transfected cells were cultured in the indicated medium for 6 hours. Total cell extracts (T), 1% Triton X-100-soluble (S) and Triton X-100-insoluble (I) fractions were prepared as described in the Materials and Methods. In E-G, anti-Flag immunoprecipitation products were subjected to immunoblotting with anti-Flag or anti-myc. All results shown are representative and were reproduced at least twice.