7C). temperature on the shaker. Plates were washed, and anti-IgG2b HRP-conjugated secondary (Southern Biotech) diluted 1 : 1000 in 5% milk/TBS was added to each well and incubated for 1 h at room temperature. After washing, plates were developed with Horseradish Peroxidase Substrate Kit (Biorad). Color was allowed to develop for 15 min. Plates were read with an Infinite m200 plate reader (Tecan) at 405 nm. Phospho-Tau ELISA Flat-bottomed 96 well plates were coated with purified anti-tau antibody (DA31) (2 g/mL) [36] in coating buffer overnight at 4C. Plates were blocked with Starting Block (Pierce) for 90 min at room heat with shaking. Samples (= 4 per timepoint) were diluted in 20% Superblock (Pierce) in TBS, added to the plate, and serial diluted then incubated overnight at 4C with shaking. After washing, either 4G10 anti-phosphotyrosine (Millipore) or biotinylated DA9, diluted in 20% Superblock (Pierce) -TBS was added to the plate and incubated for 1 h at room temperature. After washing, HRP conjugated anti-IgG2b or streptavidin-HRP (Southern Biotech), diluted 1 : 1000 in 5% Milk/TBS was added to the plate and allowed to incubate for 1 h at room temperature. Plates were washed and TMB-Ultra (Pierce) Auristatin F was added to each well for 20 min. TMB reaction was stopped with 4N H2SO4. Plates were read with an Infinite m200 plate reader (Tecan) at 450 nm. Biotinylation of DA9 was peformed using EZ-link NHS-PEO solid phase biotinylation kit (Pierce). Statistical analysis One-way ANOVA with Dunnetts post-test was performed using Graphpad Prism. RESULTS Transgene expression is doxycycline dependent and forebrain-specific in AblPP/tTA mice Forebrain-specific neuronal expression of constitutively active c-Abl (AblPP), consistent with Auristatin F CamKIIpromoter expression, occurred in AblPP/tTA mice (Fig. 1A, B). Abl-PP appeared to be confined to the neuronal cytoplasm, and was not found in nucleus (Fig. 1B). There was dense neuritic staining, in addition to staining of neuronal cell bodies. There was no significant AblPP expression in the cerebellum or brainstem, and no expression of the AblPP transgene was detectable in the absence of the CamKII= 4 per timepoint. One-way ANOVA with Dunnetts post-test was used to calculate significance of Abltide phosphorylation in AblPP/tTA mice vs. single transgenic (tTA) controls. * 0.05, ** 0.01, ***= 4 for each timepoint. One-way ANOVA with Dunnetts post-test was used to calculate whether AblPP/tTA mice showed significant increases in tau phosphorylation Auristatin F when compared Tg to control (tTA) mice. * 0.05, ** 0.01, *** 0.001. CCE) PHF1 staining of representative sections of AblPP/tTA mouse CA1 (D, E) versus controls (C). Scale bars = 800 m (C, D), 400 m (E). ArgPP/tTA mice do not exhibit the same pathological phenotype as AblPP/tTA mice, despite comparable levels of transgene expression and kinase activity We created a second line of transgenic mice expressing a constitutively active form of the Arg protein (ArgPP), the other member of the Abl family of kinases, under the forebrain specific CamKIIpromoter and the inducible tet-off system. We refer to this line of mice as ArgPP/tTA Auristatin F mice. Densitometry measurements of Western blots of cortex homogenates of AblPP/tTA and ArgPP/tTA with the AR23 antibody, an antibody with equal affinity for both Abl and Arg proteins, showed that there was no significant difference in protein expression in the cortex of the two lines of mice at both 12 and 24 weeks off doxycycline (Fig. 7A, B). Additionally, Abl kinase assay analysis showed that both lines of transgenic mice had.