na k atpase α3  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: Early Developmental Characteristics and Features of a Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

doi: 10.3390/ijms25158203

Figure Lengend Snippet: Representative images of immunofluorescence for retinoschisin (RS1) and Na/K-ATPase in control and patient retinal organoids (ROs) at day 90 and 120 of differentiation. ( A ) Immunofluorescence staining of RS1 (green) and retinal Na/K-ATPase subunits α3 (ATP1A3) (red) in the outer layer of in the outer layer of control and patient ROs at days 90 and 120 (scale bar = 50 µm). ( B ) Immunofluorescence staining of RS1 (green) and retinal Na/K-ATPase subunits β2 (ATP1B2) (red) in the outer layer of control and patient ROs at days 90 and 120 (scale bar = 50 µm).

Article Snippet: The primary antibodies used were: RS1 (1:1000; Abcam, Cambridge, UK), RCVRN (1:100; Millipore, Burlington, MA), Na/K-ATPase α3 (1:500; Almone Labs, Jerusalem, Israel), Na/K-ATPase β2 (1:500; Novus Biologicals, Centennial, CO, USA), phospho-p44/42 MAPK (phosphor-Erk1/2) (1:500; Cell Signaling Technology, Danvers, MA, USA), CRX (1:1000; Abnova, Taipei, Taiwan), and NRL (1:1000; R&D, Minneapolis, MN, USA).

Techniques: Immunofluorescence, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Early Developmental Characteristics and Features of a Three-Dimensional Retinal Organoid Model of X-Linked Juvenile Retinoschisis

doi: 10.3390/ijms25158203

Figure Lengend Snippet: Effect of co-culture of control and X-linked juvenile retinoschisis patient retinal organoids (ROs). ( A ) Western blot analysis of the expression of the retinoschisin (RS1) protein in the culture medium of control ROs at days 90. ( B ) Quantitative analysis of band density. Each experiment is executed in triplicate. Data are shown as mean ± standard error ( n = 3; **** p < 0.001, one-way analysis of variance). ( C ) Co-culture experiment involving control ROs and patient ROs using co-culture plate. (Gray circle: Control ROs, Black circle: Patient ROs, Arrow: Medium flow direction) ( D ) Immunofluorescence staining of RS1 (green) and photoreceptor marker recoverin (RCVRN) (red) in patient ROs following co-culture (scale bar = 50 µm). ( E ) Immunofluorescence staining of RS1 (green) and retinal Na/K-ATPase subunits α3 (ATP1A3) (red) in patient ROs following co-culture (scale bar = 50 µm). ( F ) Immunofluorescence staining of RS1 (green) and retinal Na/K-ATPase subunits β2 (ATP1B2) (red) in patient ROs following co-culture (scale bar = 50 µm). ( G ) Immunofluorescence staining of p-44/42 (green) in patient ROs following co-culture (scale bar = 50 µm).

Article Snippet: The primary antibodies used were: RS1 (1:1000; Abcam, Cambridge, UK), RCVRN (1:100; Millipore, Burlington, MA), Na/K-ATPase α3 (1:500; Almone Labs, Jerusalem, Israel), Na/K-ATPase β2 (1:500; Novus Biologicals, Centennial, CO, USA), phospho-p44/42 MAPK (phosphor-Erk1/2) (1:500; Cell Signaling Technology, Danvers, MA, USA), CRX (1:1000; Abnova, Taipei, Taiwan), and NRL (1:1000; R&D, Minneapolis, MN, USA).

Techniques: Co-Culture Assay, Control, Western Blot, Expressing, Immunofluorescence, Staining, Marker

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: ASPD inhibit ex vivo relaxation response of blood vessels through NAKα3 inhibition (A–C) The effect of ASPD (with or without 2-hr preincubation with ASPD-specific mASD3 antibody) in A or ouabain in C on the carbachol dose-dependent induction of the relaxation response of phenylephrine-constricted ex vivo rat aortic rings. The rat isolated aortic rings were treated with ASPD, ASPD preincubated with mASD3 antibody (0.1 mg/mL) ( Noguchi et al., 2009 ; Ohnishi et al., 2015 ), or ouabain (an inhibitor for rodent NAKα3 at the concentration used) at the indicated concentrations and the carbachol-induced relaxation response was examined by monitoring the isometric tension change (see “ ”). Data are expressed as a percentage to the maximal constriction induced by phenylephrine (n = 5, except for mASD3-preincubated ASPD (n = 3)). In vitro -reconstituted synthetic ASPD, which share essential characteristics with patient-derived ASPD (see Table S1 in ( Ohnishi et al., 2015 )), were used generally, except for the experiments in Figure 7 . ED 50 and ED 10 values ofcarbachol required for relaxation are shown below the plots. Data are presented as means ± S.E. ∗∗ P < 0.01 (ANOVA with Scheffé’s method (A) and Welch’s t test (C)). (B) Double immunofluorescence staining of rat aortic rings prepared as in A was performed with antibodies specific for NAKα3 and vWF (see “ ”). The arrows indicate NAKα3 on the apical surface of the endothelium. Scale bars: 1 μm.

Article Snippet: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques: Ex Vivo, Inhibition, Isolation, Concentration Assay, In Vitro, Derivative Assay, Double Immunofluorescence Staining

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: Binding target of ASPD on human brain microvessel endothelial cells is NAKα3 (A) Primary human brain microvessel endothelial cells were stained with NAKα3-specific antibody and DAPI nuclear stain, and high-power fluorescence images were acquired using a confocal laser-scanning microscope LSM710 with a x100 oil-immersion objective lens (see “ ”). Representative 2D images are shown on the left and the vertical section image obtained from the ZStack 3D images is shown on the right. Scale bars: 5 μm. (B) NAKα3 expression in the cells in A was determined by Western blotting using a NAKα3-specific antibody (left panels) or by RT-PCR analysis of ATP1A3 mRNA (right panels) (see “ ”). For Western blotting, 70 μg and 5 μg proteins were loaded for endothelial cells and primary neurons, respectively (see “ ”). (C) Endothelial cells treated with ASPD at the indicated concentration for 10 min were fixed and stained with ASPD-specific mASD3 antibody ( Noguchi et al., 2009 ), NAKα3-specific antibody, and DAPI nuclear stain (see “ ”). The fluorescence 2D images were captured using a confocal quantitative image cytometer CQ1 (left panels), and the ratio of ASPD-bound NAKα3 to total NAKα3 was obtained as a Mander correlation coefficient using CQ1 software (right, n = 5) (see “ ”). Scale bars: 5 μm. Data are presented as means ± S.E. (D) High-power 2D fluorescence images of the cells treated with ASPD (30 nM) in C were acquired as in A (upper). The vertical section image on the lower left and the line-scan analysis of the fluorescence intensity of NAKα3 (green line) or ASPD (red line) on the lower right were obtained from the ZStack 3D images of the same cells using Zen2009 software (see “ ”). Scale bars: 5 μm for solid line and 1 μm for hatched line. (E) The expression levels of NAKα3 in the cells without transfection or with ATP1A3 siRNA (s1724, Thermo Fisher Scientific) or MOCK siRNA (negative control, 4390843, Thermo Fisher Scientific) transfection for 3 days were determined by Western blotting (left) and by quantification of NAKα3 staining (right) (see “ ”). Data in the lower left are shown as the ratio of NAKα3 to actin, and the ratio for the non-treated cells is shown as 100 (n = 3) (see “ ”). 2D immunofluorescence images of NAKα3 staining as shown on the left were captured as in C. The NAKα3 number/cell on the right was calculated by dividing the number of punctate NAKα3 stains by the number of DAPI stains using CQ1 software (n = 5) (see “ ”). Scale bars: 5 μm. Data are presented as means ± S.E. ∗ P < 0.05 (ANOVA with Scheffé’s method). (F) Endothelial cells, without transfection, or with ATP1A3 siRNA or MOCK siRNA transfection as in E, were treated with ASPD (30 nM) for 10 min, and were stained with specific antibodies as in C. High-power 2D fluorescence images of the cells were obtained as in A (left panels). The ratio of the ASPD-bound NAKα3 to total NAKα3 was obtained as a Mander correlation coefficient, as in C (right, n = 5). Scale bars: 5 μm. Data are presented as means ± S.E. ∗ P < 0.05 (ANOVA with Scheffé’s method).

Article Snippet: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques: Binding Assay, Staining, Fluorescence, Laser-Scanning Microscopy, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Concentration Assay, Cytometry, Software, Transfection, Negative Control, Immunofluorescence

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: Comparison of caveolin-1-binding motifs in human NAKα1 and NAKα3

Article Snippet: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques:

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: ASPD-NAKα3 interaction in caveolae increases the phosphorylation of eNOS-Thr 495 in primary human cerebral endothelial cells (A) Primary human brain microvessel endothelial cells, treated with ASPD at the indicated concentration for 0, 10, or 60 min, were multiply stained with the indicated antibodies; NAKα3-specific antibody, eNOS-specific antibody, lipid rafts flotillin-1-specific antibody, and ASPD-specific antibody (rabbit polyclonal rpASD1), along with DAPI nuclear stain, were used as described in “ ”. The weighted colocalization coefficients were obtained using ZEN2009 software (the eNOS-overlapped NAKα3/total NAKα3 ratios in the cells treated with ASPD for 0, 10, and 60 min are 55.7 ± 2.4%, 58.8 ± 4.8%, and 56.0 ± 2.4%, respectively; the NAKα3-overlapped eNOS/total eNOS ratios in the cells treated with ASPD for 0, 10, and 60 min are 40.8 ± 1.4%, 42.7 ± 2.1%, and 40.1 ± 1.7%, respectively; the flotillin-1-overlapped NAKα3/total NAKα3 ratios in the cells treated with ASPD for 0, 10, and 60 min are 4.5 ± 0.9%, 4.9 ± 1.2%, and 3.6 ± 0.9%, respectively: data are presented as means ± S.E. (n = 10)) (see “ ”). The weighted colocalization coefficients represent the number of red (or green) pixels that co-localize with green (or red) pixels divided by the total number of red (or green) pixels. Scale bars: 5 μm for solid line and 1 μm for hatched line. (B) Schematic illustration of the relationship between the NO production and the phosphorylation at Ser 1177 /Thr 495 of eNOS. Carbachol activates eNOS by inducing phosphorylation at Ser 1177 and dephosphorylation at Thr 495 in parallel (green arrows). ASPD increase Thr 495 phosphorylation of eNOS (red arrow) through an independent pathway from that of carbachol. (C) Primary human endothelial cells were treated with ASPD (35 nM) for 6 hr (see “ ”). The levels of eNOS-P-Ser 1177 , eNOS-P-Thr 495 , and eNOS-total were determined by Western blotting of total extracts with antibodies specific for eNOS-P-Ser 1177 , eNOS-P-Thr 495 , and eNOS-total, respectively, as shown in upper Western blots (see “ ”). Quantification data were determined by densitometry using LAS-4000 Mini software and are shown as the ratio of eNOS-P-Ser 1177 or eNOS-P-Thr 495 to eNOS-total. The ratio of non-treated cells is shown as 100 (n = 4). Data are presented as means ± S.E. (Welch’s t -test). (D) The endothelial cells were pretreated with ASPD (32 nM) for 6 hr and were further treated with carbachol (1 μM) for 5 min (see “ ”). The ratio of eNOS-P-Ser 1177 to eNOS-total was obtained and is shown as in C (n = 4). Data are presented as means ± S.E. ∗ P < 0.05/∗∗ P < 0.01 (ANOVA with Scheffé’s method). (E) The endothelial cells, with ATP1A3 siRNA or Mock siRNA transfection or without transfection as in Figure 3 E, were treated with ASPD (32 nM) for 6 hr. The ratio of eNOS-P-Thr 495 to eNOS-total was obtained as in C (n = 3). Data are presented as means ± S.E. ∗ P < 0.05/∗∗ P < 0.01 (ANOVA with Scheffé’s method).

Article Snippet: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques: Concentration Assay, Staining, Software, De-Phosphorylation Assay, Western Blot, Transfection

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet: Schematic illustration of the mechanism of ASPD-induced suppression of eNOS activity in brain microvessel endothelial cells ASPD bind to cell-surface NAKα3 in caveolae 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: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques: Activity Assay

Journal: iScience

Article Title: Alzheimer's Aβ assembly binds sodium pump and blocks endothelial NOS activity via ROS-PKC pathway in brain vascular endothelial cells

doi: 10.1016/j.isci.2021.102936

Figure Lengend Snippet:

Article Snippet: After the incubation, the sections were further incubated 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), and then incubated with the appropriate secondary antibodies (see above).

Techniques: Recombinant, Avidin-Biotin Assay, Synthesized, Plasmid Preparation, Bicinchoninic Acid Protein Assay, Derivative Assay, Software, Light Microscopy, Laser-Scanning Microscopy, Imaging, Cytometry