Fig. 1a. Western blot analysis for total and phospho-c-Abl isoforms in human hippocampus. Fresh frozen hippocampus from non-neurologic disease control and neuropathologically confirmed early- and late-stage AD cases were homogenized as described in Methods to generate soluble and insoluble fractions. A) Equal amounts of protein from each case were loaded in gel lanes and immunolabeled using antibodies specific to total or phospho-c-Abl. The blot was also probed with actin as a loading control for both soluble and insoluble fractions. Lane 1–5 represent control subjects, lanes 5-10 from early-stage AD subjects, and lane 11–15 are late-stage AD subjects. Top panel: soluble fraction; bottom panel: insoluble fraction.
Fig. 1b. B) Quantitative measurements of total and phospho-c-Abl in control and AD subjects, normalized by loading control. Protein level was shown as mean ± SE, n = 5 for each group. The highest protein level among all 15 cases was set to 1. Statistical analysis was performed by Kruskal-Wallis test (non-parametric one way ANOVA) across all subject groups (ANOVA P value indicated in the figure and considered significant when P < 0.05), followed by post-hoc test to compare each pair of groups (marked by * when p < 0.05 between the indicated groups).

The distribution of Abl-pY412 in control, early AD and late AD hippocampus. Representative immunohistochemistry of Abl-pY412 in the CA3 (Left column) and CA1 (Right column), for healthy control (A and B), early stage AD (C and D), and late stage AD (E and F). Magnification for all panels is 200X, and inset magnification is 400X. Abl-pY412 was observed both a granular staining and GVDs in neuronal cell bodies as indicated by arrows (insets in E and F). (Colours are visible in the electronic version of the article at www.iospress.nl)

Abl-pT735 immunolabels neuropathologic hallmarks of AD. A) Abl-pT735 immunostains plaques and NFTs (magnification: 200X). B) Abl-pT735 localizes to GVDs (magnification: 400X). C) Abl-pT735 localizes to Hirano bodies (magnification: 400X). (Colours are visible in the electronic version of the article at www.iospress.nl)

Confocal microscopy of Abl-pY412 and phospho-tau in CA1 late stage AD. (Magnification: 400X) Top panel: Abl-pY412 co-localized with phospho-tau (labeled by AT8) in GVDs within pyramidal hippocampal neurons (arrow in merged image). Bottom panel: Abl-pY412 co-localized with phospho-tau (labeled by AT8) in punctate structures within AT8 positive neuritic plaques (arrow in merged image). (green: Abl-pY412, red: p-tau, yellow: merged).

c-Abl interacts with p-tau in AD brain. Co-immunoprecipitation performed using hippocampal tissue extracts from healthy control or late AD subjects. A): Proteins were immunoprecipitated with antibody to c-Abl and proteins separated by SDS-PAGE. The resulting blot was probed with p-tau antibody (AT8). Lane 1: AD subject, lane 2: healthy control subject, lanes 3 and 4: No antibody negative controls (no antibody used for immunoprecipitation). B): Proteins were immunoprecipitated using anti-phospho-tau antibody and the resulting blot probed with antibody to c-Abl. Lane 1: AD subject, lane 2: healthy control subject, lanes 3 and 4: No antibody negative controls.

The subcellular distribution of c-Abl in control, early- and late AD hippocampus. The expression of total c-Abl in the CA3 (Left column) and CA1 (Right column) from healthy controls (A and B), early stage AD (C and D), and late stage AD (E and F) as determined using antibody specific to non-phosphorylated c-Abl (see Methods). Magnification for all panels is 200X, and inset magnification is 400X. (Colours are visible in the electronic version of the article at www.iospress.nl)

The distribution of Abl-pT735 in control, early AD, and late AD hippocampus. Immunohistochemistry of Abl-pT735 in the CA3 (Left column) and CA1 (Right column) of healthy controls (A and B), early stage AD (C and D), and late stage AD (E and F). Magnification for all panels is 200X and inset magnification is 400X. (Colours are visible in the electronic version of the article at www.iospress.nl)

Quantification of immunohistochemistry for total c-Abl and phosphorylated isoforms of c-Abl in control and AD hippocampus. Quantification of total c-Abl immunoreactivity (panel A), Abl-pY412 immunoreactivity (panel B) and Abl-pT735 immunoreactivity (panel C). Error bars represent standard error of each group. White bars denote the healthy control group, and the black and grey bars denote the early- and late-stage AD respectively. Statistical analysis was performed by Kruskal-Wallis test (non-parametric one way ANOVA) across all subject groups (ANOVA P value indicated in the figure and considered significant when P < 0.05.), followed by post-hoc test to compare each pair of groups (marked by * when p < 0.05 between the indicated groups).

Confocal microscopy of Abl-pT735 with tau and Aβ in late stage AD. Panels A-F): Double-label confocal microscopy for Abl-pT735 and p-tau (AT8). (Magnification: 600X) Abl-pT735 co-localizes with NFTs (indicated by arrow) but not p-tau positive dystrophic neurites (indicated by arrowhead) (panels A-C). P-tau and Abl-pT735 co-localize to GVDs (indicated by arrow, panels D-F). (green, panel A & D: Abl-pT735, red, panel B & E: AT8, yellow, panel C & F: merged). Panels G-I): Double-label confocal microscopy for Abl-pT735 and Aβ. Punctate Abl-pT735 immunoreactivity is contained within amyloid plaques, although Abl-pT735 is not co-localized with Aβ. (panel G: Abl-pT735 in green; panel H: Aβ in red; panel I: merged image with co-localization in yellow. Amyloid plaque indicated by arrow).