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Fig. 6. Depletion of cholesterol perturbs PrPT182A raft association and increases its scrapie-like conversion. (A) Sucrose density gradients. FRT cells expressing PrPT182A were grown on 150 mm dishes in control conditions (control) or after cholesterol depletion (Mev/ß-CD). After lysis in 1% Triton X-100, 2 mg of total proteins were run through a two-step (5-30%) sucrose density gradient, as described in Materials and Methods. Twelve fractions were collected from the top to bottom of the tube after centrifugation to equilibrium, and PrPT182A was revealed in each fraction by western blotting. (B) Pulse-chase analysis in cholesterol-depleted cells. FRT cells expressing PrPT182A were cholesterol depleted (Mev/ß-CD) or untreated (control). After pulse-labelling with [³⁵S]methionine for 20 minutes, they were chased in medium containing unlabelled methionine for the indicated times. Cells were then lysed and PrPT182A was immunoprecipitated and analysed by SDS-PAGE and phosphorimaging (left). The amount of protein was quantified by NIH image software and expressed as a percentage of the protein amount rescued after pulse (chase time 0) and plotted as a function of the chase time (right). The data points were fitted to an exponential curve using a nonlinear regression analysis. Squares, control samples; circles, Mev/ß-CD-treated samples. (C) PK digestion assay in cholesterol-depleted cells. PrPT182A-transfected FRT cells were grown in control (control) or cholesterol depleting (Mev/ß-CD) conditions. Cells were lysed in Triton/DOC buffer, in the absence of protease inhibitors, and were treated with PK (+) for 2 minutes. After SDS-PAGE, the PrPT182A mutant was revealed by western blotting. Data from different experiments were quantified using NIH image software for Macintosh as indicated in the results.

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