QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

Help viewing high resolution images Return to article

Fig. 5. Proteasome block does not lead to cytosolic accumulation of PrPT182A nor to an increase in PK-resistant scrapie-like forms. (A) Immunofluorescence. FRT cells expressing PrPT182A were treated with ALLN for 7 hours, fixed and permeabilized with 0.075% saponin. Then, they were treated with the -PrP mAb (PRI308) and with primary polyclonal antibodies against CNX and giantin and with -mouse and -rabbit secondary antibodies conjugated with TRITC or FITC. The samples were then examined using a Zeiss laser scanning confocal microscope (LSM 510). Cytosol/membrane fractionation. (B) FRT cells expressing PrPT182A were treated or not with ALLN and cytosol (C)/membrane (M) separation was performed as described in Materials and Methods. Proteins were TCA precipitated and PrP molecules were revealed by western blotting. (C) After cytosol (C)/membrane (M) separation, PrPT182A was immunoprecipitated and revealed by SDS-PAGE and western blotting using either -PrP (SAF32) or -polyubiquitin antibodies (left and middle panels). As total control, 1/3 of total lysates was TCA precipitated and blotted by using the anti-polyubiquitin antibody (right panel). (D) Proteinase K (PK) digestion assay. Control and ALLN-treated cells were lysed and proteins were digested (+) or not (–) with PK, as described in Materials and Methods. TCA precipitation of PrP molecules were revealed by SDS-PAGE and western blotting. Note that the ratio between the amount of total proteins loaded in PK untreated/PK treated samples is 1:3.

Return to article