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Fig. 1. Accumulation of proteinase K resistant PrP in the nuclei of prion-infected cells independently of proteasome inhibition. (A) N2a cells were either untreated or treated with 150 µM ALLN for 12 hours and processed for PrP immunofluorescence detection with anti-PrP antibodies. (B) N2a and ScN2a cells were either untreated or treated for 12 hours with 150 µM ALLN. Nuclei were isolated and analyzed by western blots with anti-PrP antibodies. (C) Nuclei extracts or total cell lysates were treated with proteinase K (16 µg/mg protein) for 30 minutes at 37°C. Samples were run on the same gel, but the lane corresponding to the nuclei sample was exposed longer than the total cell lysate lane. (D) Absence of contamination of the nuclei preparations with endoplasmic reticulum fractions was tested with anti-calnexin antibodies. The control lane C represents a total cellular lysate from N2a cells. (E) Nuclei extracts were untreated (–) or treated (+) with N-glycosidase F (PGNase) (0.01 units/ml) for 16 hours at 37°C before analysis by western blot with anti-PrP antibodies.
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