Biochemical characterization of membrane- and nucleus-associated PrP^c isoforms.

<p>(A): Stability of membrane and nuclear PrP^c was analyzed by western blot after treatment of the cells with cycloheximide for the indicated times and purification of membranes and nuclei. Bands obtained in western blots (SAF 32 antibody) were quantified by scanning densitometry. E-cadherin and PARP were used to normalize the values obtained in membrane and nuclear fractions respectively, since both proteins were found stable for the duration of CHX treatment. Histograms correspond to the ratio (%) between PrP^c and E-cadherin or PARP from the corresponding scanned bands at each time (mean±SD from 3 independent experiments), the value obtained at time 0 being set at 100. (B): To determine the glycosylation state, rafts and nuclear extracts were treated (+) or not (−) with endo F and PrP^c was analyzed by western blot (SAF 32 antibody). Molecular weight in KDa are indicated (C): The presence of a GPI anchor was analyzed after immunoprecipitation of PrP^c from rafts or nuclear extracts, SDS-PAGE, transfer and overlay with biotinylated pro-aerolysin bacterial toxin. To check the purity of the extracts, the expression of calnexin (membrane marker) and PARP (nuclear marker) was analyzed by western blot. Molecular weight in KDa are indicated.