JCS02768 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. After cholesterol depletion, the PrPT182A mutant remains localized mainly in the ER. FRT cells expressing PrPT182A were grown under cholesterol depleting conditions on coverslips, then fixed and permeabilized with 0.075% saponin. They were then incubated with the a-PrP mAb (PRI308) and with primary polyclonal antibodies against different markers of intracellular compartments [calnexin (CNX), giantin and early endosomal antigen 1 (EEA1)]. Subsequently, they were treated with a-mouse and a-rabbit secondary antibodies conjugated with TRITC or FITC. Lysotracker (lysoT) was used to label lysosomes for 1 hour in vivo before fixation and confocal imaging. The samples were examined in a Zeiss laser scanning confocal microscope (LSCM 510). Bar,10 mm.

• Supplemental Figure 2 -

  Fig. S2. The PrPT182A mutant is degraded in the lysosomal compartment. (A) 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 a combined treatment with 30 mM NH₄Cl and 200 mg/ml leupeptin (NH₄Cl+leupeptin) to inhibit lysosomal proteolytic activities (Caughey et al., 1991; Taraboulos et al., 1992), which was maintained for the entire length of the experiment. Following lysis, PrPT182A was immunoprecipitated and analysed by SDS-PAGE and phosphorimager scanning. (B) The amount of protein was quantified using NIH image software and expressed as a percentage of the amount of protein recovered after pulse (chase time 0) and plotted as a function of the chase time. Note that the half-life of PrPT182A increased from 60 minutes in control conditions, to about 120 minutes after lysosomal activity block. Squares, control samples; circles, (NH₄Cl+leupeptin)-treated samples.