F BH (10 in PBS, 5 Sarkosyl) were digested with PK (Sigma-Aldrich, St. Louis, MO, USA) in 20 mM Tris-HCl pH 8.5 at 37uC for 1 h unless otherwise stated. Naringin Digestion was stopped by addition of Pefabloc (Fluka, Buchs, Switzerland) to a final concentration of 2 mM. Deglycosylation was carried out with 2 ml of PNGase F solution (New England Biolabs, Ipswich, MA, USA) at 37uC for 48 h, according to the manufacturer’s instructions.Digestion with PK After Partial Unfolding with Guanidinehcl (Gnd)Samples of BH (5 ml) were mixed with an equal volume of an appropriate aqueous Gnd solution to yield the desired final Gnd concentration and then incubated at 37uC for 1 h. After incubating, the samples were diluted with buffer (20 mM TrisHCl pH 8.5) to yield a 0.4 M Gnd solution, which were then treated with PK (25 mg/ml) for 1 h at 37uC. The digestion was stopped by adding Pefabloc (2 mM final concentration) and the protein was precipitated by addition of ice-cold methanol (85 final concentration). The resulting pellets were resuspended in 9 ml of deionized water, and deglycosylated with PNGase F (vide supra).Materials and Methods Ethics StatementAnimal experiments were carried out in accordance with the European Union Council Directive 86/609/EEC. The procedures and animal care were governed by a protocol that was approved by the Institutional Ethics Committee of the University of Santiago de Compostela. All efforts were made to minimize the suffering of the animals.Tricine-SDS-PAGE and Western Blot AnalysisThe precipitated pellets were boiled for 10 minutes in 10 ml of Tricine sample buffer (BioRad, Hercules, CA, USA) containing 2 (v/v) of b-mercaptoethanol. Electrophoresis was performed using precast 10?0 Tris-Tricine/Peptide gels (BioRad, Hercules, CA, USA), in the Criterion System (BioRad, Hercules, CA, USA). The cathode buffer was Tris-Tricine-SDS buffer 1 6 (Sigma-Aldrich, St. Louis, MO, USA) and the anode buffer, 1 M Tris-HCl pH 8.9. Electrophoresis was performed at constant voltage (125 volts) for 200 minutes, on ice. The gels were electroblotted (350 mA, for 150 minutes; 4uC) onto PVDF membranes (Immobilon-P, 0.45 mm; Millipore, Billerica, MA, USA). Membranes were probed with the following monoclonal antibodies: mAb #51 (epitope: G92-K100), undiluted; W226 (epitope: W144-N152), at 1:5000 dilution; or R1 (epitope: Y225-S230), at a 1:5000 dilution. Peroxidase-conjugated anti-mouse or anti-human antibodies (GE Healthcare, Little Chalfont, UK)AnimalsTransgenic heterozygous GPI-anchorless (GPI-) 1326631 PrP mice (tg44(+/2)) were a generous gift from Bruce Chesebro, Rocky Mountain Laboratories, NIH, Montana, USA. Mice were crossed to obtain homozygous GPI- animals (tg442/2), which were identified by tail DNA analysis using the PCR protocol described by Chesebro et al. [15]. Homozygous animals were bred and expression of GPI- PrP confirmed by Western blot (Figure S1). Female mice were intracerebrally inoculated at six weeks of age with 20 ml of a 2 RML-infected mouse brain homogenate (BH), ?kindly provided by Juan Maria Torres, CISA, Madrid, Spain. After 365 days post inoculation, the asymptomatic mice [16] were euthanized, their brains surgically removed, rinsed in PBS, and stored at 280uC until needed.Structural Organization of Mammalian Prionswere used as a secondary antibody, as appropriate (1:5000 dilution). Blots were developed with ECL-plus reagent (GE Healthcare, Little Chalfont, UK). Three sets of partially overlapping MW markers, Peptide Mol.