Figure 4

Western immunoblot analysis of the PMCA products from cattle brains. Western immunoblot PrP^res signature of the PMCA products. Treatment A (PK digestion protocol for atypical PrP^res digestion). PrP^res signature changes from atypical (lanes 1 and 3) to a classical 3 band pattern (lanes 2, 4–9) after PMCA amplification in Bov-Tg110 substrate. Treatment B (higher PK concentration for PMCA amplification). PrP^res signature of the PMCA amplification products in the Bov-Tg110 substrate (lanes 11, 13–19) is indistinguishable from the BSE profile after PMCA amplification (lane 10). Atypical PrP^res (lanes 10 and 12) was not detected by WB when using this digestion protocol. Deglycosylation treatment with PNGaseF. The non-glycosylated PrP^res of the atypical scrapie PMCA amplification products (lanes 21, 23–28) have a molecular mass of 19 kDa, identical to the non-glycosylated PrP^res BSE control (lane 29). Arrows point to PrP^res bands: (1) diglycosylated band, (2) monoglycosylated band and (3) non-glycosylated band. The bands observed above the diglycosylated band (arrow 1) may represent aggregates of PrP^res generated by PMCA, the intensity of which are reduced after the deglycosylation treatment. 12B2 1/4000, Sha31 1/5000. Molecular mass markers in kilodaltons (kDa) are indicated on the sides of the blots.