Western blot analysis of dephosphorylated extracts obtained from dissected tissues of 2-month old mice using the Tau5 antibody.

<p>(<b>A</b>) Analysis of wild-type (WT) mice; brain (bra), pancreas (pan), liver (liv), kidney (kid), muscle (mus), spleen (spl), testis (tes), and heart (hea). (<b>B</b>) Inclusion of tau knock-out (KO) tissue. Molecular weight and isoforms are indicated. (<b>C</b>) Western blot analysis of dephosphorylated brain extracts from 2-month old, 2-week old and P0 WT mice using the 3R- and 4R-specific antibodies RD3 and RD4, respectively. Note: The relatively intensities of RD3 and RD4 cannot be used to deduce the relative levels of the 3R and 4R isoforms. Instead, the Tau5 pattern is informative.</p

Western blot analysis of dephosphorylated samples derived from different brain areas of 2-month old WT mice using the Tau5 antibody.

<p>(<b>A</b>) Brain tissues: cortex (ctx), hippocampus (hip), pituitary gland (pit), striatum (str), cerebellum (cer) and olfactory bulb (olf). (<b>B</b>) Relative levels of tau isoforms in the different brain areas. (<b>C</b>) Significance analysis using two-way ANOVA. The significance level is calculated by comparing to cortex. *, P< 0.05, **, P< 0.01, ***, P< 0.001, and ns, not significant. Error bars represent the standard error of the mean (SEM).</p

Additional file 1: Figure S1. of A local insult of okadaic acid in wild-type mice induces tau phosphorylation and protein aggregation in anatomically distinct brain regions

Detection sensitivity of the OA ELISA. A) In water, OA is detectable down to spiked concentrations of 0.075 ng, with little background signal in the water only control. B) As positive control, homogenized brain was spiked with 100 ng OA, a 10x higher dose than used experimentally. OA is detected at significant levels in positively spiked tissue compared to non-spiked brain homogenate. A slight background signal is detected in the non-spiked samples suggesting low levels of non-specific binding in brain tissue compared to water (n = 3, **p < 0.01). Figure S2. Total protein content assessed by the sum of protein concentration from all fractions per animal reveals no significant difference between RAB fractions. At 24 h, OA induces a small but significant increase in the insoluble RIPA fraction which persists, but is no longer significant by 7 days (n = 3, *p < 0.05). Figure S3. Western blotting quantification at 24 h assessing A,D) soluble total tau, B,E) AT180-phosphorylated tau, and C,F) the phospho-tau/total tau ratio. Figure S4. Quantification of fractions at 7 days, observing no significant differences in trend or mean for the RAB fractions (A,B,C). D) Insoluble total tau levels are non-significantly elevated by OA in the posterior quadrants. E) OA-treatment significantly increases AT180 phosphorylation in both injected and contralateral posterior quadrants. F) Overall trend for increased phospho/total tau ratio in the soluble fractions for all quadrants (n = 3, *p < 0.05). Figure S5. Multiple tau residues are hyperphosphorylated in the insoluble fraction at 7 days. Representative blots from RIPA fractions probed for total tau (DAKO) and phosphospecific antibodies A) AT8, B) S422, C) AT270, D) S235, and E) S262. All phospho-antibodies show the most prominent increase in phospho-tau in the OA-injected PI fraction (see also the additional higher molecular weight signal). F) No significant differences were detected with a nitrated tau-specific antibody. (PDF 1020 kb

Immunohistochemical analysis of tau isoforms in 2 month-old wild-type mice.

<p>(<b>A</b>-<b>D</b>) Testing of antibodies reveals no bleach-through. Staining of the hippocampus with (<b>A</b>) Tau5 and (<b>C</b>) Dako tau. Omitting the primary antibodies and reacting the sections only with the secondary antibodies (<b>B</b>) Alexa Fluor 555 goat anti-mouse IgG, or (<b>D</b>) Alexa Fluor 488 goat anti-rabbit IgG. (<b>E</b>-<b>H</b>) Pre-absorption with the peptides, with which the corresponding antibodies pan-tau M (<b>E</b>), 0N (<b>F</b>), 1N (<b>G</b>) and 2N (<b>H</b>) were generated. (<b>I</b>-<b>L</b>) Staining with the new antibodies M (<b>I</b>), 0N (<b>J</b>), 1N (<b>K</b>) and 2N (<b>L</b>) in red, (<b>M</b>-<b>P</b>) counter-staining with Dako tau in green, (<b>Q</b>-<b>T</b>) Merged images. (<b>U</b>-<b>X</b>) Sections from tau knock-out mice used as negative control for antibodies pan-tau M (<b>U</b>), 0N (<b>V</b>), 1N (<b>W</b>) and 2N (<b>X</b>). Scale bar: 50 μm.</p

Subcellular fractionation of brains from 2-month old WT mice.

<p>(<b>A</b>) Relative purity of the cytoplasmic (C), membrane (M), nuclear (N), chromatin-bound (nuclear) (Ch) and cytoskeletal (S) fractions were confirmed using the following antibodies: GAPDH (for C), GLT-1 (for M), hnRNP-1 (for N), histone H2A (for Ch), and GFAP (for S). (<b>B</b>) Western blot analysis of dephosphorylated brain fractions obtained from 2-month old WT mice using the tau-specific Tau5 antibody. (<b>C</b>) Relative ratio of the three tau isoforms in the five fractions. (<b>D</b>) Significance analysis using two-way ANOVA. The significance level is calculated by comparing to the cytoplasmic fraction. *, P< 0.05, **, P< 0.01, ***, P< 0.001, and ns, not significant. Error bars represent the standard error of the mean (SEM).</p

Raising tau isoform-specific antibodies.

<p>(<b>A</b>) Schematic representation of the exon structure of the MAPT locus that encodes murine tau. Alternative splicing of exons 2, 3, and 10 generates the three isoforms 0N4R, 1N4R, and 2N4R that are present in the adult murine brain. The scheme shows the location of the epitopes that were used to raise specific antibodies for 0N, 1N and 2N murine tau, as well as for total murine tau (M), without cross-reactivity with human tau. (<b>B</b>) Western blot analysis of RAB-soluble tau extracts obtained from brains of 2-months old wild-type (WT) mice, with stripes probed separately with Tau5, M (total mouse tau) and the murine tau isoform-specific antibodies 01, 2N, and 2N reveals their specificity. Tau knock-out (KO) tissue was included as negative control.</p

Expression of murine tau isoforms in the hippocampus of WT mice at day P0.

<p>At this stage, 0N (0N3R) is the major isoform. (<b>A</b>-<b>D</b>) Staining with the pan-tau M antibody (<b>A</b>), 0N (<b>B</b>), 1N (<b>C</b>), and 2N (<b>D</b>). (<b>E</b>-<b>H</b>) Counter-staining with Dako tau. Scale bar: 50 μm.</p

Subcellular fractionation of brains from WT mice at P0.

<p>At this stage, 4R isoforms are not expressed. 0N3R is the predominant species, and 2N3R is not detected. (<b>A</b>) Western blot analysis using the same subcellular markers as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084849#pone-0084849-g004" target="_blank">Figure 4</a>. (<b>B</b>) Western blot analysis of dephosphorylated brain fractions obtained from P0 WT mice using Tau5. (<b>C</b>) Relative ratio of the three tau isoforms in the fractions. (<b>D</b>) Significance analysis using two-way ANOVA. </p

Relative ratio of tau isoforms in five subcellular fractions at P0, 2 weeks and 2 months of age.

<p>(<b>A</b>) Cytoplasmic, (<b>B</b>) membrane, (<b>C</b>) soluble nuclear, (<b>D</b>) chromatin-bound, and (<b>E</b>) cytoskeletal fraction. Error bars represent the standard error of the mean (SEM).</p

Downward gradient in S6rp phosphorylation levels in CINs along the dorsomedial-to-ventrolateral axis.

<p>(A) Coronal mouse brain diagrams at the levels analyzed illustrating a red-to-white color gradient from the most dorsomedial point of the striatum (dashed arrow). (B) Quantification of p-rpS6 in individual CINs (color-coded dots) and their distance to the most dorsomedial point. (N = 2,107 neurons, 4 mice). (C) Spatial mapping of CINs color-coded according to their relative p-rpS6 fluorescence levels in representative sections of the anterior (top) and posterior (bottom) striatum.</p