Figure 5

Human PrP displays similar PK resistance and aggregation ability after tunicamycin treatment. RK13 cells stably expressing wild-type PrP, V180I, N197D, or N181D/N197D were cultured for 7 days; tunicamycin was then added to the culture medium 48 h before the cells were harvested. Wild-type PrP and its mutants in RK13 cells treated with tunicamycin were digested with various concentrations of PK (from left to right, 0, 0.5, 1.0, 2.0, 4.0, and 8.0 ng/μl) and probed with the anti-PrP antibody 3F4 (a). The normalized amounts of insoluble PrP aggregates in the stable cells treated with tunicamycin (c) were calculated as the ratio of the density of insoluble PrP aggregate bands probed by 3F4 to that of the total PrP bands in cell lysates also probed by 3F4 (b). RK13 cells stably expressing wild-type PrP were used as a control. The gel and blot images of wild-type PrP, V180I, N197D, and N181D/N197D (a,b) shown in the figure were cropped from different parts of the same gel with an exposure time of 30 s. Clear delineation with white spaces is used. Full-length blots and gels are presented in Supplementary Figure 6.