Figure 3.

Chronically infected N2a/Bos2 cells “cured” of PrP^Sc by antibody 6H4 treatment continue to produce PrP and are susceptible to reinfection. (a) Chronically infected N2a/Bos2 cells, treated with antibody 6H4 for 2 weeks at the concentrations indicated and propagated in its absence for 66 days were not exposed (upper 2 rows) or exposed to 0.1% RML-infected mouse brain homogenate for 3 days (lower 2 rows). After culturing for 14 days, PrP^Sc was monitored by the cell blot assay. (b) Relative susceptibility to prions of N2a/Bos2 cells (BOS2) and N2a/Bos2 cells “cured” by exposure to antibody 6H4 at 20 μg/ml was determined by exposing cultures to various dilutions of RML-infected mouse brain homogenate for 3 days and determining PrP^Sc as above. (c) Levels of PrP^C and PrP^Sc in various sublines were determined by Western blotting. Chronically infected N2a/Bos2 cells, treated for 2 weeks with antibody 6H4 at the concentrations indicated, were passaged for 84 days after antibody withdrawal. Cells were lysed and samples corresponding to 2.25 × 10⁵ cells were incubated in the presence (+PK) or absence (−PK) of proteinase K (5 μg/ml) for 90 min at 37°C. Western blotting was performed as described in Materials and Methods. UN, uninfected N2a/Bos2 cells; I-BOS2, chronically prion-infected BOS2 cells. Molecular mass markers are indicated at the left of each panel.