Fig. 4

Western blot analyses for total and phosphorylated tau. Equal amounts of proteins from detergent-soluble brain lysates of 20-month-old triple-transgenic Alzheimer’s disease (3xTg-AD) mice (D1–D5 = DNA Aβ₄₂-immunized mice, P1–P4 = amyloid-β 1–42 [Aβ₄₂] peptide-immunized mice, C1–C5 = 3xTg-AD control mice, wt = wild-type controls) were separated by SDS-PAGE, blotted onto nitrocellulose filters, and probed using antibodies specific for total human tau (a, upper panel), and phosphorylated tau AT8 (a, middle panel), and β-tubulin as a loading control (a, bottom panel). The graph on the right-hand side of the SDS-PAGE pictures shows analyses of the band intensities performed with ImageJ software. All gray-level intensities of tau protein bands were normalized to the gray-level intensities of protein bands of the housekeeping proteins β-tubulin or β-actin, respectively. The reduction of total tau in the DNA-immunized mice compared with the 3xTg-AD control animals was highly significant (p = 0.0059). Of note, gray-level intensities for sample D2 were not included in thess calculations, because the loading control for this sample indicated a much lower protein content (a, bottom panel). b A comparison of total tau levels in DNA-immunized mice, 3xTg-AD control mice, and wt control mice in Western blots is shown using two different antibodies. In the upper panel, 43D (Tau1–100) was used for detection; in the middle panel, antibody HT7 was used; and in the lower panel, the same membrane was probed with a β-actin antibody as a protein loading control. The graph on the right-hand side of the panels shows the analyses of gray-level intensities for the protein bands with ImageJ software normalized to gray-level intensities of the housekeeping protein β-actin. Differences were statistically significant with p values of 0.0152 (HT7) and 0.0138 (43D). * and ** indicate p values of ≤ 0.05 and ≤ 0.01, respectively (Mann-Whitney U test)