Fig. 2. PrPwt and T182A mutant acquire different oligosaccharide modifications in FRT cells. (A) FRT cells expressing PrPwt and PrPT182A mutant were lysed for 20 minutes in Triton/DOC buffer. PrPs were either untreated (control lanes) or digested for 16 hours with 5 mU/sample Endo-H (lanes H) or neuraminidase (lanes N). After TCA precipitation, proteins were revealed by SDS-PAGE and western blot with PRI308 antibody and chemiluminescence. D, diglycosylated PrP; M, monoglycosylated PrP; U, unglycosylated PrP. (B) FRT cells expressing PrPT182A were pulse-labelled with [³⁵S]methionine for 20 minutes and then chased in medium containing unlabeled methionine for the indicated times. Cells were then lysed in Triton/DOC buffer and PrPT182A was immunoprecipitated. Half of the samples were treated with Endo-H (+) and half was left untreated (–) prior to analysis by SDS-PAGE and phosphorimager scanning.