H status, while neurologic status was assessed three times per week. At the first appearance of symptoms, mice were weighed prior to each neurological exam. Animals were euthanized when they showed signs of progressive neurological dysfunction or had lost 20 of their initial weight. One sagittal half of each brain was fixed in 10 formalin, while the other half was immediately frozen and stored at 280uC until processed for immunoblotting (see below). One-way ANOVA was used to test whether there were any significant differences in average time to onset of paresis or survival following inoculation.ImmunoblottingMouse brain Calcitonin (salmon) web hemispheres were homogenized in 1.0 ml EMBO buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA 1 Triton X) per 0.1 g of brain weight. Whole protein was quantified using bicinchoninic acid assay (Pierce). For PrPSc detection, samples were digested with proteinase K (1 mg/50 mg total protein) for 1 h at 37 C, followed by addition of PMSF (2 mM final concentration). Samples (20 mg of total protein) were subjected to electrophoresis 223488-57-1 through 10 or 14 Novex TrisGlycine gels (Invitrogen) and transferred to PVDF membranes. Blots were blocked with 5 non-fat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h, incubated with anti-prion antibody D18 (0.5ug/mL) overnight at 4 C, then washed and incubated at room temperature for 2 hrs with HRP-conjugated goat anti-human (Bio-Rad) secondary antibody (1:5000 in 1 notfat dry milk) before being developed using ECL Western Blotting Substrate (Thermo Scientific Pierce).HistologyFormalin-fixed samples were processed, paraffin-embedded, and sectioned at 8 mm thickness. Sections of septum, hippocampus/thalamus, midbrain and cerebellum/brainstem were stained with hematoxylin and eosin (H E, Fisher Scientific SH26?00D and 245?58) or processed by immunohistochemistry for PrP (R2 monoclonal antibody, a gift from Dr. Stanley Prusiner) or the astrocyte marker, GFAP (Dako #Z0334). The secondary antibody for R2 was biotinylated 22948146 goat anti-human Kappa chain (Vector Laboratories #BA?060) and for GFAP was biotinylated goat anti-rabbit IgG (Vector Laboratories #BA?000). The Peroxidase substrate DAB kit (Vector Laboratories #SK?100) was used for color development. The percentage area occupied by vacuoles in a high power field was measured on H E stained sections from 2 animals of each genotype. Each brain region was scored on one section for each animal. A score of 0 indicates no vacuoles were present. A score of up to 5 could reflect a small amount of real vacuolation or artifact and is considered to represent `no significant lesion’ (NSL). Scores of 5?9, 20?9 and 70 indicate mild, moderate and severe vacuolation, respectively [15].ResultsFigure 2. Brain PrPC and PrPSc expression. (A-B) Brain protein lysates from uninoculated (A) and inoculated (B) transgenic and nontransgenic Mgrn1+/+, Mgrn1md2nc/+ and/or Mgrn1md2nc/md2nc mice were subjected to immunoblotting with an antibody against PrP. The `no PK’ panel shows all PrP species present. The protease-resistant (proteinase K treated, `PK’) panel shows that PrPSc is present in samples from all RML prion-inoculated animals but not in uninoculated animals, regardless of Mgrn1 genotype. doi:10.1371/journal.pone.0055575.gOne possible mechanism by which prion replication may cause disease is by inducing misdirection of PrP to the cytoplasm [11]. Since Mgrn1 over-expression rescued the endo-lysosomal trafficking defects associated with the pre.H status, while neurologic status was assessed three times per week. At the first appearance of symptoms, mice were weighed prior to each neurological exam. Animals were euthanized when they showed signs of progressive neurological dysfunction or had lost 20 of their initial weight. One sagittal half of each brain was fixed in 10 formalin, while the other half was immediately frozen and stored at 280uC until processed for immunoblotting (see below). One-way ANOVA was used to test whether there were any significant differences in average time to onset of paresis or survival following inoculation.ImmunoblottingMouse brain hemispheres were homogenized in 1.0 ml EMBO buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA 1 Triton X) per 0.1 g of brain weight. Whole protein was quantified using bicinchoninic acid assay (Pierce). For PrPSc detection, samples were digested with proteinase K (1 mg/50 mg total protein) for 1 h at 37 C, followed by addition of PMSF (2 mM final concentration). Samples (20 mg of total protein) were subjected to electrophoresis through 10 or 14 Novex TrisGlycine gels (Invitrogen) and transferred to PVDF membranes. Blots were blocked with 5 non-fat dry milk in Tris-buffered saline with Tween-20 (TBST) for 1 h, incubated with anti-prion antibody D18 (0.5ug/mL) overnight at 4 C, then washed and incubated at room temperature for 2 hrs with HRP-conjugated goat anti-human (Bio-Rad) secondary antibody (1:5000 in 1 notfat dry milk) before being developed using ECL Western Blotting Substrate (Thermo Scientific Pierce).HistologyFormalin-fixed samples were processed, paraffin-embedded, and sectioned at 8 mm thickness. Sections of septum, hippocampus/thalamus, midbrain and cerebellum/brainstem were stained with hematoxylin and eosin (H E, Fisher Scientific SH26?00D and 245?58) or processed by immunohistochemistry for PrP (R2 monoclonal antibody, a gift from Dr. Stanley Prusiner) or the astrocyte marker, GFAP (Dako #Z0334). The secondary antibody for R2 was biotinylated 22948146 goat anti-human Kappa chain (Vector Laboratories #BA?060) and for GFAP was biotinylated goat anti-rabbit IgG (Vector Laboratories #BA?000). The Peroxidase substrate DAB kit (Vector Laboratories #SK?100) was used for color development. The percentage area occupied by vacuoles in a high power field was measured on H E stained sections from 2 animals of each genotype. Each brain region was scored on one section for each animal. A score of 0 indicates no vacuoles were present. A score of up to 5 could reflect a small amount of real vacuolation or artifact and is considered to represent `no significant lesion’ (NSL). Scores of 5?9, 20?9 and 70 indicate mild, moderate and severe vacuolation, respectively [15].ResultsFigure 2. Brain PrPC and PrPSc expression. (A-B) Brain protein lysates from uninoculated (A) and inoculated (B) transgenic and nontransgenic Mgrn1+/+, Mgrn1md2nc/+ and/or Mgrn1md2nc/md2nc mice were subjected to immunoblotting with an antibody against PrP. The `no PK’ panel shows all PrP species present. The protease-resistant (proteinase K treated, `PK’) panel shows that PrPSc is present in samples from all RML prion-inoculated animals but not in uninoculated animals, regardless of Mgrn1 genotype. doi:10.1371/journal.pone.0055575.gOne possible mechanism by which prion replication may cause disease is by inducing misdirection of PrP to the cytoplasm [11]. Since Mgrn1 over-expression rescued the endo-lysosomal trafficking defects associated with the pre.
