Fig. 4. Long-term effect of Prnp-specific siRNA on the PrP-res accumulation. N2aS12sc⁺ cells were transfected without (0) or with 400 nM Prnp-specific siRNA duplex. At confluence, cells were lysed and one-tenth of the samples were analysed in the absence of PK-treatment (–PK), whereas the remaining samples were PK-digested (+PK). These samples are labeled as 0 passage. Alternatively, cells were cultured for 1 and 2 weeks with splitting 1:4 every 4 days, i.e. 2 and 4 passages, respectively. PrP-sen and PrP-res levels were then detected by immunoblotting with SAF83 monoclonal antibodies as described in Materials and Methods. Molecular mass markers in kilodaltons (kDa) are indicated on the left.