Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A,C: Effect of 46 nM ASPD (A) or 100 nM ouabain (C) on carbachol-induced relaxation of ex-vivo rat aortic rings (n = 3 for mASD3-preincubated ASPD-treated group, and n = 5 for other groups). B: Representative images of immunohistochemical multiple staining of NAKα3 and vWF in the endothelial layer of rat aortic rings. The arrowheads indicate NAKα3 on the apical surface of endothelium. Scale bars: 1 μm. In (A,C), data are presented as means ± S.E. ** P < 0.01 (ANOVA with Scheffé’s method (A) and Welch’s t -test (C)).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Ex Vivo, Immunohistochemical staining, Staining

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: A: Representative 2D images of immunocytochemical staining of NAKα3 and nuclei (DAPI) on human primary brain microvessel endothelial cells (left) and vertical section image prepared from z-stack 3D image (right). Scale bars: 5 μm. B: Western blotting for NAKα3 in the endothelial cells (left) and agarose electrophoresis of RT-PCR products for ATP1A3 mRNA (right). C: Binding ratio of ASPD signal to total NAKα3 signal (the representative images in upper panels and the quantification in bottom, n = 5). Scale bars: 5 μm. D: High-power representative 2D images of multiple immunocytochemical staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated endothelial cells (left). The vertical section image prepared from z-stack 3D image (right upper) and its line-scan analysis of fluorescence intensities of NAKα3 (green line) and ASPD (red line) (right bottom). Scale bars: 5 μm for solid line and 1 μm for hatched line. E: Western blotting for NAKα3 in the siRNA-transfected endothelial cells (left, n = 3) and quantification of number of punctate NAKα3 signal on the endothelial cells (right, n = 5). Scale bars: 5 μm. F: Effect of siRNA transfection on the binding ratio of ASPD signal to total NAKα3 signal on the endothelial cells (n = 5). The transfection of ATP1A3 siRNA decreased the ASPD binding ratio to 37 ± 12%, 11 ± 2%, 42 ± 2%, and 66.2 ± 2% in the 3 nM, 10 nM, 30 nM, and 100 nM ASPD-treated groups, respectively. In (C,E,F), data are presented as means ± S.E. * P < 0.05 (ANOVA with Scheffé’s method).

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Western Blot, Electrophoresis, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Fluorescence, Transfection

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: High-power representative 2D images of immunocytochemical multiple staining of ASPD (ASPD-specific, mASD3 antibody), NAKα3, and nuclei (DAPI) on 30 nM ASPD-treated human primary brain microvessel endothelial cells with or without siRNA transfection (Upper, middle, and bottom panels show non-treated, mock siRNA-treated, and ATP1A3 siRNA-treated cells, respectively). Scale bars: 5 μm.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Staining, Transfection

Journal: bioRxiv

Article Title: Alzheimer’s Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway

doi: 10.1101/2020.12.10.419879

Figure Lengend Snippet: ASPD bind to cell-surface NAKα3 on cerebral microvessel endothelial cells, promote mitochondrial ROS production, activate PKC, increase eNOS-Thr 495 phosphorylation, and attenuate NO release, resulting in suppression of blood microvessel relaxation response.

Article Snippet: These sections were incubated overnight at 4°C with primary antibody against NAKα3 (ANP-003, 1:200; Alomone Labs, Jerusalem, Israel) and von Willebrand factor glycoprotein (sc-365712, 1:50; Santa Cruz Biotechnology, Dallas, TX) in the presence of normal goat serum in PBS, and then incubated with the appropriate Alexa Fluorconjugated secondary antibody (1:1000, Molecular Probes, Waltham, MA) for 1 hr at r.t. Counterstaining was carried out with 4’,6-diamidino-2-phenylindole (DAPI, 1:500; Dojindo Molecular Technologies, Kumamoto, Japan).

Techniques: Phospho-proteomics