Fig. 2. Proteinase K status of prion protein mutants from total cell lysates of transfected BHK cells. Cells were transiently transfected with recombinant SFV RNAs encoding wild-type human PrP (lanes 1-4), human PrP-FFI (lanes 5-8) and human PrP+9OR (lanes 9-12). 24 hours post-transfection the expression was analyzed by western blotting using mAb anti-PrP 3F4 (upper panel) and pAb W3 [directed against the laminin receptor (Gauczynski et al., 2001b); lower panel]. Crude lysates were treated with increasing amounts of proteinase K for 30 minutes at 37°C: 0 µg/ml (lanes 1, 5 and 9), 2 µg/ml (lanes 2, 6 and 10), 4 µg/ml (lanes 3, 7 and 11) and 8 µg/ml (lanes 4, 8 and 12, upper panel). Proteinase-K-resistant bands range from 23 to 25 kDa as indicated (brackets). To determine the LRP level, crude lysates were treated with increasing amounts of proteinase K buffer (in the absence of proteinase K) for 30 minutes at 37°C (lower panel).