Figure 4.
Pulse-chase analysis of PrPC after cholesterol and sphingolipid depletion. FRT cells grown in control (control), cholesterol depletion (Mev/βCD), and sphingolipid depletion (FB1) conditions (see Materials and Methods) were subjected to pulse-chase analysis by using [35S]methionine for the indicated times. At the end of each chase time the cells were lysed in Triton/Doc buffer and PrPC was immunoprecipitated with SAF 32 antibody. The immunoprecipitated material was treated (+) or not (-) with Endo-H (5 mU/sample for 16 h at 37°C). The protein was revealed by SDS-PAGE and phosphorimager scanning. Quantitation of four independent experiments are shown in the graphs in the right panels. Error bars are reported from the different quantitations. H, mature, highly glycosylated PrPC isoform; I*, unmodified diglycosylated isoform; IΔ, monoglycosylated form; U, unglycosylated form.