The comparison of hippocampus major axis length between control and experiment.

<p>Control: 7 month-old hemizygous + B6 deficient.</p><p>Experimental: 7 month-old hemizygous male +B6 deficient + anti-HA antibody.</p><p>5 different sections were observed and its major axis was measured.</p><p>Average (n = 2) is shown.</p><p>Non-Tg control showed Hippocampal major axis of 10±0.5 cm.</p

Long-Term Memory Test in the Morris Water Maze task.

<p>Non-transgenic mice had an average score of 3 mice each day. Hemizygous transgenic control (hemizygous + B6 deficient) mice had an average score of 3 transgenic control mice each day. Transgenic experimental (hemizygous + B6 deficient + anti-HA antibody) mice had an average score of 3 transgenic experimental mice each day. 3xTg-AD mice were aged 3 months and 3 weeks. Statistically significant difference was observed after 2 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (<i>P</i><0.001) and after 3 trial days between hemizygous + B6 deficient and hemizygous + B6 deficient + antibody (<i>P</i><0.05).</p

Hematoxylin-Eosin Staining of Cortical and Hippocampal Neurons in Control and Experimental Groups.

<p>Mice were treated the same as those shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#pone-0008593-g003" target="_blank">Fig. 3</a>.</p

Immunohistochemical observations of Amygdalar, Cortical, and Hippocampal Neurons.

<p>Anti-HA antibody was diluted 100-fold. Immunohistochemical observations were repeated thrice and each observation gave the same result. Ten homozygous transgenic 3-month-old mice were fed B6-deficient food for 3 weeks. Transgenic experimental mice were injected with anti-HA antibody every 3 days. For details, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. CTL, transgenic control mice; Ex, transgenic experimental mice.</p

The Effect on Memory Performance of HA Vaccine on Normal Feeding of 3xTg-AD Male Mice.

<p>Mice were 12 months old. Memory performance was measured via the Morris water experiment described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. Statistically significant difference was observed on the third, fourth, and fifth trial days (<i>P</i><0.001). 3xTg-AD male mice were all homozygous (n = 15).</p

Curative Effect of Anti-HA Antibody as shown by Long-Term Memory Performance.

<p>Seven-month-old male 3xTg-AD homozygous mice mice fed B6-deficient food for 3 weeks served as the control. Experimental mice were treated with anti-HA antibody every 3 days; antibody (100-fold dilution) was injected into the brain as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. The figure shows average data for five male mice. To demonstrate the strong curative effect of the antibody, for comparison we show the results for 2-month-old hemizygous male mice, whose memory performance was normal. Statistically significant difference was observed after 2 trial days (<i>P</i><0.001). However after 3 trial days, a statistically significant difference was not observed (^*<i>P</i><0.001). The difference between control and experiment mice (<i>P</i><0.01). The difference between experimental mice and 7-month-old mice with normal food (n = 5).</p

HA level in 3xTg-AD mice.

<p>Note: A: Vaccine treatment was started at 12 months. Three months later, urine was collected an entire day and HA level was measured. 3xTg-AD male mice were all homozygous. HA level with vaccine treatment decreased with time, indicating that vaccine efficacy became stronger with time.</p><p><b>Note: For reference, HA level in 12-month-old in 3xTg-AD male mice were observed.</b></p><p>Note: B: After Morris water maze test, these mice were sacrificed and their brains were immediately freezed in liquid nitrogen. Next, HA level was measured with the method described in Materials and Method.</p

Homocysteic Acid Level in 3xTg-AD mice brain at 4 months compared with Non-Tg control mice.

<p>HA level in the brain of 3xTg-AD male mice at 4 months was measured by the method described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>. Control was non-Tg male mice at 4 months.</p

HA Level in 3xTg-AD mice brain after Ingesting Vitamin B6-Deficient food for 3 weeks.

<p>Control mice: 3xTg-AD mice at 3 months and 3 weeks with normal feeding. Control mice showed good memory performance, but B6-deficient feeding group showed memory impairment. Whole brain was homogenized with physiological saline and HA level was measured, after the memory task, according to the method described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008593#s2" target="_blank">Materials and Methods</a>.</p

Determination of Rate-Limiting Factor for Formation of Beta-Catenin Destruction Complexes Using Absolute Protein Quantification

Wnt/β-catenin signaling plays important roles in both ontogenesis and development. In the absence of a Wnt stimulus, β-catenin is degraded by a multiprotein “destruction complex” that includes Axin, APC, GSK3B, and FBXW11. Although the key molecules required for transducing Wnt signals have been identified, a quantitative understanding of this pathway has been lacking. Here, we calculated the absolute number of β-catenin destruction complexes by absolute protein quantification using LC–MS/MS. Similar amounts of destruction complex-constituting proteins and β-catenin interacted, and the number of destruction complexes was calculated to be about 1468 molecules/cell. We demonstrated that the calculated number of destruction complexes was valid for control of the β-catenin destruction rate under steady-state conditions. Interestingly, APC had the minimum expression level among the destruction complex components at about 2233 molecules/cell, and this number approximately corresponded to the calculated number of destruction complexes. Decreased APC expression by siRNA transfection decreased the number of destruction complexes, resulting in β-catenin accumulation and stimulation of the transcriptional activity of T-cell factor. Taken together, our results suggest that the amount of APC expression is the rate-limiting factor for the constitution of β-catenin destruction complexes