Analysis of preclinical prion-inoculated EAM / control mice. a) NaPTA blots (muscle and brain) as well as regular Western blots (spleen) show PrPSc load over time in EAM versus untreated mice. As positive controls for NaPTA blots RML is used as a spike in untreated tissue homogenate and is either PK-digested or undigested. For blot of spleen brain homogenate from a terminally diseased wild type mouse is loaded with and without PK- digestion. As a negative control, PK-digested normal tissue homogenate is used. Note that there are differences in PrPSc loads in muscle at day 35 and 60 in EAM versus control mice. b) Quantifications of western blots of autophagy marker LC3 show elevated amounts of LC3 II versus LC3 I in muscle versus spleen and brain of C57/Bl6 mice at day 0. (n = 4; control = brain homogenate of a cathepsin D knockout mouse). c) H&E as well as immunohistochemical staining for CD3 show signs of myositis in the muscle tissue only in treated mice in contrast to untreated controls. In brain tissue in both cohorts no gross pathological hallmarks of prion disease could be detected by H&E and immunohistochemical staining for GFAP over time (scale bar is 200 μm and 20 μm for brain and 100 μm and 20 μm for muscle). d) Bioassays to determine titers of prion infectivity reveal no differences between both prion-inoculated EAM and prion-inoculated control mice.