Figure 1. moPrP^RL converts to a PK resistant isoform at lower concentrations than moPrP^WT.

moPrP^WT and moPrP^RL at three different concentrations were incubated in the presence (E–H) or absence (A–D) of POPG lipid vesicles. Samples were removed for proteolytic digestion by proteinase K (PK) followed by ultra-centrifugation and Western blot analysis at four different time points. Generation of protease resistant, insoluble PrP was compared over time between moPrP^WT and moPrP^RL. In POPG vesicle containing samples, generation of material that is both insoluble and resistant to PK digestion was detectible after only five minutes (E) as a 15 kDa PrP band (denoted by large arrow). A smaller molecular weight band of approximately 14.5 kDa (denoted by small arrow) was detectible by 24 hours (F). Conversion to a PK resistant, insoluble form at a concentration of 100 µg/mL is observed for only moPrP^RL, consistently at all time periods analyzed (E–H, lanes 3 vs 6). Over time, PK treated, ultracentrifuged samples accumulated a 24 kDa band (denoted by double arrow) (E–H). This band may represent undigested rPrP or a dimer of smaller molecular weight, PK cleaved fragments. Generation of aggregated, PK resistant material in the absence of lipids was not detected for either moPrP^WT or moPrP^RL (A–D). PK resistant material was also undetectable in the soluble fraction (A–H). Data shown is representative of results from four independent experiments.