All of the procedures involving live animals were approved by the Canadian Science Centre for Human and Animal Health – Animal Care Committee (CSCHAH-ACC) or the University of British Columbia Animal Care Committee according to the guidelines set by the Canadian Council on Animal Care. The approval identifications for this study were animal use document (AUD)#H-08-009 and AUD#H-11-020.
Animals and sample collection
CD1 mice (Charles River Laboratories) between 4–6 weeks of age were infected intraperitoneally with the Rocky Mountain Laboratory (RML) mouse-adapted strain of scrapie. The inoculums consisted of 200 μl of 1% brain homogenate in PBS from either clinically ill or normal control mice. Animals were sacrificed at 70, 90, 110, 130 days post infection (dpi) and terminal disease (155–190 dpi). Clinical signs depicting terminal disease consisted of kyphosis, dull ruffled coat, weight loss of 20% or more and ataxia. Brain samples were collected and macrodissected into three sections, (i) cortical, (ii) subcortical (including thalamus, hypothalamus and hippocampus) and (iii) brainstem-cerebellum. Each section was flash frozen using a dry ice/methanol mixture and stored at −80°C until processing. A total of 3 scrapie- and 3 mock-infected samples were collected per time point.
All procedures were performed at 4°C or on ice. Weighed frozen brainstem-cerebellum, subcortical, and cortical regions from mock- or scrapie-infected mice were homogenized in 3 mL of freshly prepared lysis buffer (20 mM MOPS [pH 7.0], 2 mM EGTA, 5 mM EDTA, 1% Nonidet P-40, 0.01% phosphatase inhibitor cocktail [Pierce, Rockford, Illinois, USA], 0.02% protease inhibitor cocktail [Sigma-Aldrich, St. Louis, Missouri, USA], 10 mM DTT, pH 7.2)
 per 250 mg of brain, using a tissue homogenizer with disposable tips (TH and hard tissue OMNI tip, respectively; OMNI International, Kennesaw, Georgia, USA). Brain homogenates were passed twice through a 21 gauge needle, sonicated five times for 20 s intervals at 88 W output (431C1 cup horn probe, S-4000 sonicator; Qsonica, Newtown, Connecticut, USA), and pre-cleared by centrifugation for 30 min at 14,000 × g (JLA 16.250 BC rotor, Avanti J-E centrifuge; Beckman/Coulter, Brea, California, USA). Approximately 200 μL aliquots of supernatant were aliquoted into 1.5 mL tubes, snap frozen in liquid nitrogen and stored at −80°C.
Protein concentration was determined by Bradford’s assay (Bio-Rad Laboratories, Hercules, California, USA). Protein concentration and equal sample loading was re-tested in preliminary sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Brain homogenates were mixed with an equal volume of 2X SDS loading buffer (125 mM Tris-Cl [pH 6.8], 20% glycerol, 4% SDS, 0.005% bromophenol blue, 260 mM DTT) and denatured by incubation at 100°C for 10 min. Afterward, 15-well 8% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories) were loaded with 40 μg of denatured protein per linear cm of well (running buffer; 190 mM glycine, 24.8 mM Tris, 0.1% SDS, pH 8.3). Proteins were run through the stacking gel at 50 V, and then resolved for 90 min at 100 V, always at room temperature. Proteins were stained with Coomassie blue G-250 (Bio-Safe Coomassie; Bio-Rad Laboratories) according to the manufacturer’s instructions. Signal from Coomassie-stained protein was detected using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, Nebraska, USA) in the 700 nm channel and quantitated using Odyssey 3.0 software (LI-COR Biosciences). Protein amounts were calculated relative to a pre-quantitated standard brain homogenate.
Sodium phosphotungstic acid precipitation
Sodium phosphotungstic acid (NaPTA) precipitation was adapted from
[112, 113]. One milligram of mouse brain homogenate was mixed with an equal volume of 4% sodium lauroylsarcosine (sarkosyl) in PBS. Samples were incubated for 10 min at 37°C with constant agitation (1000 rpm, Thermomixer; Eppendorf, Hamburg, Germany). A 37°C NaPTA solution (PBS, 4% NaPTA, 170 mM MgCl2, pH 7.4) was then added to a final concentration of 0.3% NaPTA. Samples were incubated for 60 min at 37°C with constant agitation (1000 rpm, Thermomixer; Eppendorf), then centrifuged at 37°C for 30 min at 16,000 × g (FA-45-18-11 rotor, 5418 microfuge; Eppendorf). Pellets were resuspended in 5 μL of 0.1% sarkosyl in PBS and digested with 20 μg of proteinase K (PK; in 0.01 M CaCl2) (Roche, Indianapolis, Indiana, USA) for 30 min at 37°C with a brief vortex after 15 min
. Digestion was stopped and PK-resistant protein was denatured by quickly adding an approximately equal volume 5X SDS loading buffer (300 mM Tris-Cl [pH 6.8], 50% glycerol, 25% β-mercaptoethanol, 10% SDS, 1% bromophenol blue), and immediately incubating at 100°C for 10 min. Samples were resolved by SDS-PAGE and analyzed by Western blot.
All procedures were performed at room temperature and all washes were performed using gentle rocking, unless otherwise indicated.
PrPres was analyzed in 1.0 mg of NaPTA-enriched, PK-treated mouse brain homogenate using 15-well 12% SDS-PAGE gels (NuPAGE Novex Bis-Tris; Life Technologies Inc., Carlsbad, California, USA). The running buffer (50 mM MOPS, 50 mM Tris, 1 mM EDTA, 0.1% SDS, pH 7.7) within the upper (cathode) chamber contained 5 mM sodium bisulfite. Proteins were run through the stacking gel at 60 V, and then resolved for 2.5 h at 120 V. Afterward, polyvinylidene fluoride (PVDF) membranes (Immuno-Blot, 0.2 μm; Bio-Rad Laboratories) were soaked in methanol for 2 min, then equilibrated in transfer buffer (190 mM glycine, 24.5 mM Tris, 10% methanol) for 20 min. Filter paper (2 sheets/membrane) were equilibrated in transfer buffer for 5 min. Proteins were transferred at 4°C for 2 h at 30 V. After transfer, membranes were dried, soaked in methanol for 2 min and washed twice for 10 min each in Tris-buffered saline (TBS; 140 mM NaCl, 3 mM KCl, 25 mM Tris, pH 7.6). Membranes were blocked for 1 h in TBST (TBS/0.1% Tween-20) with 5% milk, then probed with PrP primary antibody (clone SAF83; a kind gift from Dr. Deborah McKenzie, University of Alberta) diluted to 1:10,000 in TBST with 5% milk for 18 h at 4°C. Afterward, membranes were washed with TBST once for 5 min and thrice for 10 min each. Membranes were incubated with goat anti-mouse horseradish peroxidase (HRP)-labeled secondary antibody (Bio-Rad Laboratories) diluted to 1:40,000 in TBST with 5% milk for 1 h. Membranes were washed in TBST once for 5 min and thrice for 15 min each, incubated for 5 min with enhanced chemiluminescent substrate (SuperSignal West Femto; Pierce) and then exposed to film (Super RX; Fujifilm, Tokyo, Japan). Exposed film was developed and scanned (CanoScan LiDE 200; Canon, Tokyo, Japan). Signal was quantitated using ImageJ (Version 1.47c; National Institutes of Health, Bethesda, Maryland, USA).
To analyze GFAP and total PrP, brain homogenates were mixed with a one-fourth volume of 5X SDS loading buffer. Then, 100 μg of denatured protein was loaded per linear cm onto 15-well 12% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories). Proteins were run through the stacking gel at 50 V, and then resolved for 100 min at 100 V. Afterward, gels were equilibrated in transfer buffer (384 mM glycine, 49.6 mM Tris, 20% methanol, 0.01% SDS)
 for 30 min. PVDF membranes and filter paper (2 sheets/membrane) were also equilibrated in transfer buffer for 20 and 5 min, respectively. Proteins were transferred for 23 h at 4°C; 1 h at 54 mA, 4 h at 189 mA, 8 h at 270 mA and 10 h at 378 mA. Membranes were blocked with 10% blocking buffer (Sigma-Aldrich) for 1 h and probed simultaneously with primary antibodies specific for PrP (clone SAF83) and GFAP (Abcam Inc., Cambridge, Massachusetts, USA) diluted to 1:10,000 and 1:20,000 in 10% blocking buffer with 0.1% Tween-20, respectively, for 18 h at 4°C. Afterward, membranes were washed with TBST once for 5 min and thrice for 10 min each. Membranes were then incubated for 1 h with donkey anti-mouse IRDye 680- (LI-COR Biosciences) and donkey anti-rabbit IRDye 800- (LI-COR Biosciences) labeled secondary antibodies diluted to 1:20,000 in 10% blocking buffer with 0.1% Tween-20 and 0.01% SDS. Afterward, membranes were washed in TBST thrice for 10 min each, and once with TBS for 5 min. Signal from pre-stained protein standards and IRDye 680-labeled secondary antibody was detected at 700 nm using an Odyssey infrared imaging system (LI-COR Biosciences). Signal from IRDye 800-labeled secondary antibody was detected at 800 nm. Signal was quantitated using Odyssey 3.0 software (LI-COR Biosciences). Membranes were then stained with Coomassie blue R-250 (Bio-Rad Laboratories) for 10 min before destaining with 40% methanol in 10% glacial acetic acid thrice for 10 min each, or until excess stain was removed. Signal from Coomassie-stained protein was detected at 700 nm using the Odyssey and quantitated using Odyssey 3.0 software.
For multiplex Western blots, brain homogenates were mixed with an equal volume of 2X, or one-fifth volume of 6X (375 mM Tris-Cl [pH 6.8], 60% glycerol, 12% SDS, 0.015% bromophenol blue, 780 mM DTT), SDS-PAGE loading buffer, and then 200 μg of denatured protein was loaded per linear cm of single-well 8% SDS-PAGE gels (Mini-PROTEAN; Bio-Rad Laboratories). Proteins were resolved as described for protein quantitation and transferred as described for Western blots of GFAP and total PrP. Dried membranes were probed immediately or stored at −30°C. All multiplex Western blots were performed in three sets, each composed of one membrane from a mock- and one from a scrapie-infected mouse euthanized at 70, 90, 110, 130 dpi, or at terminal stage of disease. Membranes were blocked for 1 h with 10% blocking buffer (Sigma-Aldrich) for evaluation of total protein levels, or in 3% BSA (Rockland, Gilbertsville, Pennsylvania, USA) for evaluation of phosphorylation levels. Membranes were rinsed briefly with TBS and positioned within a 24-lane multi-screen apparatus (MPX; LI-COR Biosciences). Combinations of primary antibodies were diluted in 10% blocking buffer or 3% BSA, as appropriate, with 0.1% Tween-20. One hundred sixty microliters of each antibody dilution was loaded in each lane of the multi-screen apparatus. After incubation for 18 h at 4°C, membranes were briefly washed once with TBST within the multi-screen apparatus then removed from the apparatus and further washed in TBST, once for 5 min and four times for 15 min each. Membranes were incubated with secondary antibody diluted to 1:20,000 in 10% blocking buffer or 3% BSA, as appropriate, with 0.1% Tween-20 and 0.01% SDS for 1 h. Mouse monoclonal primary antibodies were detected with donkey anti-mouse IRDye 680-labeled secondary antibody. Rabbit or goat primary polyclonal primary antibodies were detected with donkey anti-rabbit or donkey anti-goat IRDye 800-labeled secondary antibody (LI-COR Biosciences), respectively. Membranes were washed in TBST four times for 15 min each and once with TBS for 5 min. Signal from IRDye 680- and IRDye 800-labeled secondary antibody was detected at 700 and 800 nm, respectively, using the Odyssey system. Signal was quantitated using Odyssey 3.0 software.
Membranes from scrapie-infected mice were stripped (only once) together and in parallel with the membranes from the mock-infected mice from the same set, under conditions to minimize protein loss
. Membranes were stripped with mild stripping buffer (25 mM glycine, 1% SDS, pH 2.0) once for 5 min and four times for 15 min each, then with harsh stripping buffer (50 mM Tris-Cl [pH 7.0], 2% SDS, 50 mM DTT)
 once for 15 min at 37°C. Afterward, stripped membranes were washed with TBST once and TBS once for 5 min each, then blocked and reprobed with a second set of primary antibodies as described.
Hierarchical cluster analysis
For each set, the densitometric data from the primary multiplex Western blots of brainstem-cerebellum homogenates of scrapie-infected mice was normalized to that from the mock-infected mice. Relative protein kinase expression levels were then log2 transformed and analyzed with Gene Cluster 3.0
 using Euclidean distance and complete linkage. Java Treeview was used to present the clusters
All data was analyzed using Prism (Version 5.0f; GraphPad Software Inc., La Jolla, California, USA). The targeted Western blots were performed in three experimental sets on different days. Each set encompassed one scrapie- and one mock-infected mouse from each time point. The ratios from each set were thus analyzed by paired t-test. The ratios were log transformed before analyses to transform the increased and decreased ratios (ratios greater or smaller than 1, respectively) to proportional values. For nonlinear regression analyses, curves of the normalized total and phosphorylation protein levels in scrapie-infected mice were compared to no changes (a line with intercept = 1, slope = 0), representing the levels in mock-infected mice, using a replicates test for lack-of-fit.