Initially, brain samples from tg66 mice were screened for proteinase K (PK)-resistant PrP (PrPres) by immunoblot as previously described . No bands were visualized using this technique so additional testing was performed using an adapted sodium PTA procedure  for each sample. For each experimental group, 8–9 mice were analyzed for PrPres following sodium phosphotungstic acid (PTA) precipitation. For each mouse, 20% w/v brain homogenates (BH) were made in phosphate buffered saline (PBS) using a mini-bead beater system set to homogenize for 45 s, and were stored frozen at − 20 C. For further use, homogenates were thawed and diluted in PBS to create 10% homogenates. 500 μl of a 10% BH was mixed with an equal volume of 4% Sarkosyl, vortexed, and incubated in a water bath at 37 °C for 30 min. Benzonase (5 U/μl) and magnesium chloride (0.2 M) were then added to final concentrations of 25 U/ml and 0.001 M, respectively. Samples were vortexed and incubated in a water bath at 37 °C for 45 min. Centrifugation at 5000×g for 5 min at room temperature was performed, and the supernatant was transferred to a new tube. PK was added to a final concentration of 20 μg/ml, and the mixture was vortexed and incubated in a water bath at 37 °C for 1 h. The reaction was stopped with a 5 mM final concentration of Pefabloc. Four percent sodium PTA and 34 mM magnesium chloride, pH 7.4, were added to final concentrations of 0.3% and 2.73 mM, respectively, and the solution was incubated in a water bath at 37 °C for 1 h. Samples were then centrifuged at 16,000×g for 30 min at 37 °C, and the supernatants were discarded. Pellets were then resuspended in 200 μl of PBS-EDTA (40 ml of 0.5 M EDTA and 60 ml of PBS, pH 7.4), incubated for 30 min in a 37 °C water bath, and then centrifuged at 16,000×g for 30 min at 37 °C. The supernatants were again discarded, and the pellet was resuspended in 60 μl of Laemmli sample buffer, vortexed, and boiled for 5 min. 20 μl was loaded into a single lane on a 16% Tris-glycine gel (Invitrogen, Thermo Fischer Scientific) and electrophoresed. Gels were transferred to polyvinylidene difluoride membranes with the iBlot transfer system using a 7-min transfer, program 3 (Life Technologies). Membranes were probed with a 1:3000 dilution of mouse anti-PrP antibody 3F4. The secondary antibody was peroxidase-conjugated rabbit anti-mouse IgG at 1:80,000 (Sigma), and immunoreactive bands were visualized with film using a SuperSignal West Femto (Thermo Scientific) detection system. A panel of molecular weight marker proteins was run on each gel (Bio-Rad, Kaleidoscope or SDS Low Range), and the approximate size of each PrP band was calculated by extrapolation using these markers.