qRT-PCR, immunohistochemical, and immunoblot analyses of BMP levels in the hippocampus of AD patients. To screen for changes in BMP expression levels in AD brains, mRNA was extracted from the hippocampus of age-matched nondemented control (ND ctrl) and AD brains and prepared for qRT-PCR analysis. A–C, Levels of BMP2 (A), BMP6 (B), and BMP7 (C) mRNA were measured and corrected for β-actin levels as a control. Of the three BMPs studied, only levels of BMP6 mRNA (B) were significantly increased in the brains of AD patients compared to nondemented control cases. For immunohistochemical analysis, D–F depict representative immunolabeling with a mouse monoclonal antibody against BMP6 (Millipore), and G–I show representative immunolabeling with a rabbit polyclonal antibody against BMP6 (Abgent). All images are from the dentate gyrus, and sections were costained with DAPI (blue) to label cell nuclei. Scale bar, 40 μm for all images. D–F, Immunohistochemical analysis with a monoclonal antibody against BMP6 (detected with Tyramide Red) showed that BMP6 immunoreactivity (arrows, E, F) increased in the hippocampus of AD brains compared to nondemented controls. In severe AD cases, BMP6-immunoreactive cells (arrows, F) were detected in both the subgranular zone and the granular cell layer. G–I, Immunohistochemical analysis with a polyclonal antibody against BMP6 (detected with Tyramide Red) showed a similar pattern of BMP6 immunoreactivity (arrows, H, I). J, Semiquantitative analysis of levels of BMP6 immunoreactivity by immunohistochemistry with the Millipore antibody confirmed that BMP6 protein levels were increased in the hippocampus of AD brains compared to controls. K, Representative immunoblot analysis with the Millipore antibody showing increased expression of the mature form of BMP6 (15 kDa band) in homogenates from the hippocampus of AD patients compared to nondemented controls. L, Semiquantitative analysis of levels of BMP6 as measured by immunoblot in hippocampal brain homogenates confirmed increased BMP6 immunoreactivity in the hippocampus of AD patients compared to controls (n = 5 nondemented control cases, n = 7 early AD, and n = 7 severe AD cases analyzed, *p < 0.05 and **p < 0.01 compared to nondemented controls by one-way ANOVA with post hoc Dunnett's test).