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Fig. 4. The untranslocated precursor of PrPT182A is degraded by the proteasome and ubiquitylated. (A) Steady-state proteasomal block. FRT cells expressing PrPwt and PrPT182A were treated with ALLN (150 µM) for 7 hours (+). The cells were then lysed and blotted with an 
-PrP antibody. Note that in the presence of ALLN a new PrP-specific band of 
22 kDa is visible (arrow). (B) Pulse-chase analysis. FRT cells expressing PrPT182A were pulse-labelled with [35S]methionine for 20 minutes and then chased in medium containing unlabelled methionine for the indicated times in control conditions and after ALLN treatment, which was maintained for the entire length of the experiment. After lysis, PrPT182A was immunoprecipited and analysed by SDS-PAGE and phosphorimager scanning. The amount of the protein was quantified by NIH image software and expressed as a percentage of the amount of protein rescued after pulse (chase time 0) and plotted as a function of the chase time. The data points were fitted to an exponential curve using a non-linear regression analysis. Squares: control samples; circles: ALLN-treated samples. (C) PK protection assay. Microsomes were prepared as described in Materials and Methods and either left untreated or incubated with 250 µg/ml PK in the absence or presence of Triton X-100. The proteins were TCA precipitated and PrP molecules were detected by western blotting. The arrow indicates the ALLN-induced form of PrP that is selectively digested with PK in the absence of detergent. (D) Detection of PrP ubiquitylation. Cells were lysed in denaturing conditions (see Materials and Methods) and PrPT182A was immunoprecipitated with SAF32 antibody. After reduction in ß-mercaptoethanol, the sample was split in two, run on SDS-PAGE and revealed by western blotting either with the SAF32 antibody or a specific -polyubiquitin antibody to reveal polyubiquitylated PrP molecules. (E) Duplicates of each sample shown in D were run in parallel and then subjected to another run in a second dimension in SDS-PAGE. PrPT182A was revealed by western blotting either using -prion or -polyubiquitin antibodies. M, monoglycosylated PrP; Ut, untranslocated PrP; *, immunoglobulin chains and >>, polyubiquitylated PrP.
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