Support a PrPSc structure consisting of a series of highly PK-resistant b-sheet strands interspersed with PKsensitive short flexible loops and turns. Furthermore, the region comprising ,V179 to the C-terminus of PrPSc is probably composed primarily of b-sheet, as it is highly resistant to PK. Our data are consistent with our previous results (263K and Dy strains) and those of other researchers using SHaPrPSc. Furthermore, they are consistent with those observed for human CJD PrPSc, which suggests that the myriad human, hamster and mouse prions share a common basic structure.Limited ProteolysisAliquots of 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. 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 Hexaconazole custom synthesis 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 Docosahexaenoyl ethanolamide biological activity 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-) 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]. 1407003 Homozygous animals were bred and expression of GPI- PrP confirmed by Western bl.Support a PrPSc structure consisting of a series of highly PK-resistant b-sheet strands interspersed with PKsensitive short flexible loops and turns. Furthermore, the region comprising ,V179 to the C-terminus of PrPSc is probably composed primarily of b-sheet, as it is highly resistant to PK. Our data are consistent with our previous results (263K and Dy strains) and those of other researchers using SHaPrPSc. Furthermore, they are consistent with those observed for human CJD PrPSc, which suggests that the myriad human, hamster and mouse prions share a common basic structure.Limited ProteolysisAliquots of 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. 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-) 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]. 1407003 Homozygous animals were bred and expression of GPI- PrP confirmed by Western bl.
