1 vector system (Invitrogen) according to the manufacturer’s instructions. After verifying the respective specificities of the cDNA clones by sequencing, these were used to generate individual standard curves, thus allowing for calculation of molarity and number of mRNA molecules in the samples.
Finally, the respective tau mRNA levels were normalized to murine tau mRNA levels. Animals were sacrificed by decapitation, the brains were extracted, and either the brain or hippocampi BVD-523 chemical structure and EC were dissected. Tissue was homogenized in radio-immunoprecipitation assay buffer (Sigma) supplemented with a cocktail of protease and phosphatase inhibitors (Roche). Samples were homogenized using a Polytron and stored at −80°C. The materials for SDS-PAGE were obtained from Invitrogen (NuPAGE system). Protein lysates were boiled in sample buffer consisting of lithium dodecyl sulfate sample buffer and reducing agent
and resolved on 4%–12% Bis-Tris polyacrylamide precast gels in a 3-(n-morpholino)propanesulfonic acid-SDS running buffer containing antioxidant. For most analyses, 30 μg/lane were loaded, unless indicated otherwise. Gels were transferred onto Nitrocellulose Membranes Protran (Whatman) in transfer buffer containing 20% methanol. Blots were blocked in Odyssey blocking buffer (Li-Cor biosciences), followed by incubation with primary antibodies (β -actin [Sigma; 1:10,000]; Total Tau [Dako; 1:10,000], HT7 [1:5,000], TauY9 [1;1,1000], mTau [Naruhiko Sahara; 1:5,000], AT180 [pT231, Thermo Scientific; 1:1,000], Wnt cancer PHF1 [courtesy of Peter Davies; 1:5,000], CP13 [courtesy of Peter Davies, 1:1,000], and DA9 [courtesy of Peter Davies; 1: 10,000]) and detected with anti-mouse or anti-rabbit IgG conjugated to IRDye 680 or 800 (Li-Cor Biosciences;
1:10,000). Densitometric and MW analyses were performed using ImageJ software (National Institutes of Health). Band density values were normalized to β-actin or total tau levels when tau phosphorylation levels were analyzed. Mean band densities for samples Linifanib (ABT-869) from rTgTauEC mice were normalized to corresponding samples from control mice. Purification of sarkosyl-insoluble tau was performed as previously described (Hasegawa et al., 2007) with slight modifications. Briefly, whole frozen brains of 24- and 18-month-old rTgTauEC (n = 3), control (n = 3), and 18-month-old rTg4510 (n = 1) mice were homogenized by polytron in 10 volumes of buffer H (10 mM Tris-HCl [pH 7.5] containing 0.8M NaCl, 1 mM EGTA, and 1 mM dithiothreitol) and spun at 100,000 × g for 30 min at 4°C. Another 2 ml of buffer H was added to the pellet and the samples were homogenized again by polytron, incubated in 1% Triton X-100 at 37°C for 30 min.