Factor H promotes early prion propagation in spleen at early timepoints Mice (n ≥ 6 mice/genotype of both sexes) were sacrificed at 30, 60, 90 dpi, and 120 dpi, and their spleens (10% w/v homogenates) were first treated with proteinase K (PK, 10 μg/mL, panel A). Lines to the left of each blot mark 25 kD molecular weight. RML infected animals negative for PrP Sc after straight western blotting (A) were subjected to serial rounds of PMCA (B,C). Asterisks indicate samples scored as positive for PrP Sc . Each biological replicate was run in technical duplicates, and each lane represents one mouse. Western blot analysis of PMCA material (B) shows a higher proportion of RML-infected wild type mice (solid line) positive by round 1 of PMCA, (top blot), whereas most knockout mice (bottom blot, dotted line) required 2 or more rounds of PMCA to visualize PrP Sc . RML (far right) represents 0.05% RML5 amplified prions and serves as a positive control for each PMCA round. ANOVA analysis revealed no significant differences at 30 dpi, but significant differences between the genotypes at 60, 90, and 120 dpi ((p=0.0236, 0.0021, and 0.0032, respectively). Samples in first lane of each blot were not PK digested. Numbers > 100 rpu in the grey area in graph (C) represent scores from samples detected without amplification, and therefore fall outside the dynamic range of PMCA.
{}