asic1a  (Alomone Labs)


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    Alomone Labs asic1a
    Analysis of σ -1 receptor binding to <t>ASIC1a.</t> ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.
    Asic1a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asic1a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    asic1a - by Bioz Stars, 2022-08
    93/100 stars

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    1) Product Images from "Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels"

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    Journal: Biophysical Journal

    doi: 10.1016/j.bpj.2009.12.4293

    Analysis of σ -1 receptor binding to ASIC1a. ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.
    Figure Legend Snippet: Analysis of σ -1 receptor binding to ASIC1a. ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Techniques Used: Binding Assay

    AFM imaging of ASIC1a, anti-His 6 antibodies, and σ -1 receptors. ( A ) Low-magnification image of isolated ASIC1a. The arrow indicates a trimeric cluster of three equally sized particles, which is likely an ASIC trimer that has bound to the mica intact and then fallen apart during the drying process. A zoomed image of this cluster is shown at the bottom right of the panel. A shade-height scale is shown at the right. ( B ) Sections through the two particles indicated in A , taken at the positions indicated by the lines. The sections indicate that the shapes of the particles approximate that of a spherical cap. The calculated volumes of the particles are indicated. ( C ) Low-magnification image of anti-His 6 antibodies. A shade-height scale is shown at the right. ( D ) Frequency distribution of molecular volumes of anti-His 6 antibodies. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. ( E ) Low-magnification image of σ -1 receptors. A shade-height scale is shown at the right. ( F ) Frequency distribution of molecular volumes of σ -1 receptors. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.
    Figure Legend Snippet: AFM imaging of ASIC1a, anti-His 6 antibodies, and σ -1 receptors. ( A ) Low-magnification image of isolated ASIC1a. The arrow indicates a trimeric cluster of three equally sized particles, which is likely an ASIC trimer that has bound to the mica intact and then fallen apart during the drying process. A zoomed image of this cluster is shown at the bottom right of the panel. A shade-height scale is shown at the right. ( B ) Sections through the two particles indicated in A , taken at the positions indicated by the lines. The sections indicate that the shapes of the particles approximate that of a spherical cap. The calculated volumes of the particles are indicated. ( C ) Low-magnification image of anti-His 6 antibodies. A shade-height scale is shown at the right. ( D ) Frequency distribution of molecular volumes of anti-His 6 antibodies. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. ( E ) Low-magnification image of σ -1 receptors. A shade-height scale is shown at the right. ( F ) Frequency distribution of molecular volumes of σ -1 receptors. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.

    Techniques Used: Imaging, Isolation

    Analysis of anti-His 6 antibody binding to His 8 -tagged ASIC1a. ( A ) Low-magnification AFM images of a sample of isolated ASIC1a that had been incubated with anti-His 6 antibody. Arrowheads indicate singly decorated ASICs; arrows indicate doubly decorated ASICs. A shade-height scale is shown at the right. ( B ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) anti-His 6 antibodies. Lines indicate the angles between pairs of bound antibodies. ( C–E ) Frequency distributions of ( C ) molecular volumes of decorated central particles, ( D ) molecular volumes of bound peripheral particles, and ( E ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.
    Figure Legend Snippet: Analysis of anti-His 6 antibody binding to His 8 -tagged ASIC1a. ( A ) Low-magnification AFM images of a sample of isolated ASIC1a that had been incubated with anti-His 6 antibody. Arrowheads indicate singly decorated ASICs; arrows indicate doubly decorated ASICs. A shade-height scale is shown at the right. ( B ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) anti-His 6 antibodies. Lines indicate the angles between pairs of bound antibodies. ( C–E ) Frequency distributions of ( C ) molecular volumes of decorated central particles, ( D ) molecular volumes of bound peripheral particles, and ( E ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Techniques Used: Binding Assay, Isolation, Incubation

    Isolation of proteins from ASIC1a-expressing cells. ( A ) Immunofluorescence detection of ASIC1a in stably transfected HEK-293 cells. Cells were fixed, permeabilized, and incubated with either rabbit polyclonal anti-ASIC1a antibody or mouse monoclonal anti-His 6 antibody, followed by Cy3-conjugated goat anti-rabbit or anti-mouse secondary antibodies, as appropriate. Cells were imaged by confocal laser scanning microscopy. ( B ) Immunofluorescence detection of the σ -1 receptor in HEK-293 cells stably expressing ASIC1a and transiently transfected with σ -1 receptor cDNA. Cells were incubated with mouse monoclonal anti-FLAG antibody, followed by fluorescein isothiocynate-conjugated goat anti-mouse secondary antibodies. A representative immunofluorescence image is shown, along with a corresponding brightfield image; ∼20% of the cells are expressing the σ -1 receptor. ( C ) Detection of ASIC1a in a membrane fraction from stably transfected cells (M) and after isolation (I) by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using rabbit polyclonal anti-ASIC1a antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). ( D ) Detection of ASIC1a and the σ -1 receptor after isolation from σ -1 receptor-transfected cells by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using anti-ASIC1a antibody or mouse monoclonal anti-FLAG antibody. ( E ) A screen for the presence of endogenous σ -1 receptor in protein samples isolated from nontransfected ASIC1a-expressing cells or σ -1 receptor-transfected cells. Samples were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody, or rabbit polyclonal anti- σ -1 receptor antibody.
    Figure Legend Snippet: Isolation of proteins from ASIC1a-expressing cells. ( A ) Immunofluorescence detection of ASIC1a in stably transfected HEK-293 cells. Cells were fixed, permeabilized, and incubated with either rabbit polyclonal anti-ASIC1a antibody or mouse monoclonal anti-His 6 antibody, followed by Cy3-conjugated goat anti-rabbit or anti-mouse secondary antibodies, as appropriate. Cells were imaged by confocal laser scanning microscopy. ( B ) Immunofluorescence detection of the σ -1 receptor in HEK-293 cells stably expressing ASIC1a and transiently transfected with σ -1 receptor cDNA. Cells were incubated with mouse monoclonal anti-FLAG antibody, followed by fluorescein isothiocynate-conjugated goat anti-mouse secondary antibodies. A representative immunofluorescence image is shown, along with a corresponding brightfield image; ∼20% of the cells are expressing the σ -1 receptor. ( C ) Detection of ASIC1a in a membrane fraction from stably transfected cells (M) and after isolation (I) by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using rabbit polyclonal anti-ASIC1a antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). ( D ) Detection of ASIC1a and the σ -1 receptor after isolation from σ -1 receptor-transfected cells by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using anti-ASIC1a antibody or mouse monoclonal anti-FLAG antibody. ( E ) A screen for the presence of endogenous σ -1 receptor in protein samples isolated from nontransfected ASIC1a-expressing cells or σ -1 receptor-transfected cells. Samples were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody, or rabbit polyclonal anti- σ -1 receptor antibody.

    Techniques Used: Isolation, Expressing, Immunofluorescence, Stable Transfection, Transfection, Incubation, Confocal Laser Scanning Microscopy, Binding Assay, SDS Page, Silver Staining

    Determination of the raft association of ASIC1a and the σ -1 receptor. Cells were extracted with ice-cold Triton X-100 (1%) and subjected to centrifugation on Optiprep density gradients. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody (for the σ -1 receptor), or anti-caveolin antibody (as a raft marker).
    Figure Legend Snippet: Determination of the raft association of ASIC1a and the σ -1 receptor. Cells were extracted with ice-cold Triton X-100 (1%) and subjected to centrifugation on Optiprep density gradients. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody (for the σ -1 receptor), or anti-caveolin antibody (as a raft marker).

    Techniques Used: Centrifugation, SDS Page, Marker

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    Alomone Labs asic1a
    Analysis of σ -1 receptor binding to <t>ASIC1a.</t> ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.
    Asic1a, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/asic1a/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    asic1a - by Bioz Stars, 2022-08
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    Analysis of σ -1 receptor binding to ASIC1a. ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Journal: Biophysical Journal

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    doi: 10.1016/j.bpj.2009.12.4293

    Figure Lengend Snippet: Analysis of σ -1 receptor binding to ASIC1a. ( A ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) peripheral particles. Lines indicate the angles between pairs of bound peripheral particles. ( B–D ) Frequency distributions of ( B ) molecular volumes of decorated central particles, ( C ) molecular volumes of bound peripheral particles, and ( D ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Article Snippet: Proteins were detected by silver staining or immunoblotting using rabbit polyclonal antibodies against either ASIC1a (Alomone, Buckingham, UK) or the human σ -1 receptor (Abcam, Cambridge, UK), and a mouse monoclonal antibody against the FLAG tag on the σ-1 receptor (Sigma).

    Techniques: Binding Assay

    AFM imaging of ASIC1a, anti-His 6 antibodies, and σ -1 receptors. ( A ) Low-magnification image of isolated ASIC1a. The arrow indicates a trimeric cluster of three equally sized particles, which is likely an ASIC trimer that has bound to the mica intact and then fallen apart during the drying process. A zoomed image of this cluster is shown at the bottom right of the panel. A shade-height scale is shown at the right. ( B ) Sections through the two particles indicated in A , taken at the positions indicated by the lines. The sections indicate that the shapes of the particles approximate that of a spherical cap. The calculated volumes of the particles are indicated. ( C ) Low-magnification image of anti-His 6 antibodies. A shade-height scale is shown at the right. ( D ) Frequency distribution of molecular volumes of anti-His 6 antibodies. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. ( E ) Low-magnification image of σ -1 receptors. A shade-height scale is shown at the right. ( F ) Frequency distribution of molecular volumes of σ -1 receptors. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.

    Journal: Biophysical Journal

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    doi: 10.1016/j.bpj.2009.12.4293

    Figure Lengend Snippet: AFM imaging of ASIC1a, anti-His 6 antibodies, and σ -1 receptors. ( A ) Low-magnification image of isolated ASIC1a. The arrow indicates a trimeric cluster of three equally sized particles, which is likely an ASIC trimer that has bound to the mica intact and then fallen apart during the drying process. A zoomed image of this cluster is shown at the bottom right of the panel. A shade-height scale is shown at the right. ( B ) Sections through the two particles indicated in A , taken at the positions indicated by the lines. The sections indicate that the shapes of the particles approximate that of a spherical cap. The calculated volumes of the particles are indicated. ( C ) Low-magnification image of anti-His 6 antibodies. A shade-height scale is shown at the right. ( D ) Frequency distribution of molecular volumes of anti-His 6 antibodies. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated. ( E ) Low-magnification image of σ -1 receptors. A shade-height scale is shown at the right. ( F ) Frequency distribution of molecular volumes of σ -1 receptors. The curve indicates the fitted Gaussian function. The mean of the distribution is indicated.

    Article Snippet: Proteins were detected by silver staining or immunoblotting using rabbit polyclonal antibodies against either ASIC1a (Alomone, Buckingham, UK) or the human σ -1 receptor (Abcam, Cambridge, UK), and a mouse monoclonal antibody against the FLAG tag on the σ-1 receptor (Sigma).

    Techniques: Imaging, Isolation

    Analysis of anti-His 6 antibody binding to His 8 -tagged ASIC1a. ( A ) Low-magnification AFM images of a sample of isolated ASIC1a that had been incubated with anti-His 6 antibody. Arrowheads indicate singly decorated ASICs; arrows indicate doubly decorated ASICs. A shade-height scale is shown at the right. ( B ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) anti-His 6 antibodies. Lines indicate the angles between pairs of bound antibodies. ( C–E ) Frequency distributions of ( C ) molecular volumes of decorated central particles, ( D ) molecular volumes of bound peripheral particles, and ( E ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Journal: Biophysical Journal

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    doi: 10.1016/j.bpj.2009.12.4293

    Figure Lengend Snippet: Analysis of anti-His 6 antibody binding to His 8 -tagged ASIC1a. ( A ) Low-magnification AFM images of a sample of isolated ASIC1a that had been incubated with anti-His 6 antibody. Arrowheads indicate singly decorated ASICs; arrows indicate doubly decorated ASICs. A shade-height scale is shown at the right. ( B ) Gallery of zoomed images of ASICs that are undecorated ( top ) or decorated by one ( middle ) or two ( bottom ) anti-His 6 antibodies. Lines indicate the angles between pairs of bound antibodies. ( C–E ) Frequency distributions of ( C ) molecular volumes of decorated central particles, ( D ) molecular volumes of bound peripheral particles, and ( E ) angles between pairs of bound peripheral particles. The curves indicate the fitted Gaussian functions. The means of the distributions are indicated.

    Article Snippet: Proteins were detected by silver staining or immunoblotting using rabbit polyclonal antibodies against either ASIC1a (Alomone, Buckingham, UK) or the human σ -1 receptor (Abcam, Cambridge, UK), and a mouse monoclonal antibody against the FLAG tag on the σ-1 receptor (Sigma).

    Techniques: Binding Assay, Isolation, Incubation

    Isolation of proteins from ASIC1a-expressing cells. ( A ) Immunofluorescence detection of ASIC1a in stably transfected HEK-293 cells. Cells were fixed, permeabilized, and incubated with either rabbit polyclonal anti-ASIC1a antibody or mouse monoclonal anti-His 6 antibody, followed by Cy3-conjugated goat anti-rabbit or anti-mouse secondary antibodies, as appropriate. Cells were imaged by confocal laser scanning microscopy. ( B ) Immunofluorescence detection of the σ -1 receptor in HEK-293 cells stably expressing ASIC1a and transiently transfected with σ -1 receptor cDNA. Cells were incubated with mouse monoclonal anti-FLAG antibody, followed by fluorescein isothiocynate-conjugated goat anti-mouse secondary antibodies. A representative immunofluorescence image is shown, along with a corresponding brightfield image; ∼20% of the cells are expressing the σ -1 receptor. ( C ) Detection of ASIC1a in a membrane fraction from stably transfected cells (M) and after isolation (I) by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using rabbit polyclonal anti-ASIC1a antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). ( D ) Detection of ASIC1a and the σ -1 receptor after isolation from σ -1 receptor-transfected cells by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using anti-ASIC1a antibody or mouse monoclonal anti-FLAG antibody. ( E ) A screen for the presence of endogenous σ -1 receptor in protein samples isolated from nontransfected ASIC1a-expressing cells or σ -1 receptor-transfected cells. Samples were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody, or rabbit polyclonal anti- σ -1 receptor antibody.

    Journal: Biophysical Journal

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    doi: 10.1016/j.bpj.2009.12.4293

    Figure Lengend Snippet: Isolation of proteins from ASIC1a-expressing cells. ( A ) Immunofluorescence detection of ASIC1a in stably transfected HEK-293 cells. Cells were fixed, permeabilized, and incubated with either rabbit polyclonal anti-ASIC1a antibody or mouse monoclonal anti-His 6 antibody, followed by Cy3-conjugated goat anti-rabbit or anti-mouse secondary antibodies, as appropriate. Cells were imaged by confocal laser scanning microscopy. ( B ) Immunofluorescence detection of the σ -1 receptor in HEK-293 cells stably expressing ASIC1a and transiently transfected with σ -1 receptor cDNA. Cells were incubated with mouse monoclonal anti-FLAG antibody, followed by fluorescein isothiocynate-conjugated goat anti-mouse secondary antibodies. A representative immunofluorescence image is shown, along with a corresponding brightfield image; ∼20% of the cells are expressing the σ -1 receptor. ( C ) Detection of ASIC1a in a membrane fraction from stably transfected cells (M) and after isolation (I) by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using rabbit polyclonal anti-ASIC1a antibody, followed by a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody. Immunoreactive bands were visualized using enhanced chemiluminescence. Arrowheads indicate molecular mass markers (kDa). ( D ) Detection of ASIC1a and the σ -1 receptor after isolation from σ -1 receptor-transfected cells by binding to Ni 2+ -agarose. Samples were analyzed by SDS-PAGE and either silver staining ( left panel ) or immunoblotting ( right panel ) using anti-ASIC1a antibody or mouse monoclonal anti-FLAG antibody. ( E ) A screen for the presence of endogenous σ -1 receptor in protein samples isolated from nontransfected ASIC1a-expressing cells or σ -1 receptor-transfected cells. Samples were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody, or rabbit polyclonal anti- σ -1 receptor antibody.

    Article Snippet: Proteins were detected by silver staining or immunoblotting using rabbit polyclonal antibodies against either ASIC1a (Alomone, Buckingham, UK) or the human σ -1 receptor (Abcam, Cambridge, UK), and a mouse monoclonal antibody against the FLAG tag on the σ-1 receptor (Sigma).

    Techniques: Isolation, Expressing, Immunofluorescence, Stable Transfection, Transfection, Incubation, Confocal Laser Scanning Microscopy, Binding Assay, SDS Page, Silver Staining

    Determination of the raft association of ASIC1a and the σ -1 receptor. Cells were extracted with ice-cold Triton X-100 (1%) and subjected to centrifugation on Optiprep density gradients. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody (for the σ -1 receptor), or anti-caveolin antibody (as a raft marker).

    Journal: Biophysical Journal

    Article Title: Demonstration of a Direct Interaction between σ-1 Receptors and Acid-Sensing Ion Channels

    doi: 10.1016/j.bpj.2009.12.4293

    Figure Lengend Snippet: Determination of the raft association of ASIC1a and the σ -1 receptor. Cells were extracted with ice-cold Triton X-100 (1%) and subjected to centrifugation on Optiprep density gradients. Fractions from the gradient were analyzed by SDS-PAGE and immunoblotting using anti-ASIC1a antibody, anti-FLAG antibody (for the σ -1 receptor), or anti-caveolin antibody (as a raft marker).

    Article Snippet: Proteins were detected by silver staining or immunoblotting using rabbit polyclonal antibodies against either ASIC1a (Alomone, Buckingham, UK) or the human σ -1 receptor (Abcam, Cambridge, UK), and a mouse monoclonal antibody against the FLAG tag on the σ-1 receptor (Sigma).

    Techniques: Centrifugation, SDS Page, Marker

    Functional expression of ASIC2 in X. laevis oocyte. ( A ) ASIC2b coexpression attenuates ASIC2a desensitization. Original traces of whole-cell current (holding potential = –70 mV) in oocytes expressing ASIC2a alone or with ASIC2b, or t-ASIC2b, as indicated below the traces. Inward currents were induced by a rapid extracellular acidification from pH 7.4 to pH 4.0 (indicated by the horizontal bar). ( B ) Mean transient-induced (peak) and residual-induced (plateau) currents (corrected by currents recorded in control oocytes) in oocytes expressing ASIC2a alone (black bars) or coexpressing ASIC2b (red) or t-ASIC2b (blue). ( C ) Acid-induced plateau currents (normalized to maximal value achieved at pH 4) at different extracellular pH; colors as above. ( D ) Effect of amiloride: acid-induced (pH 4) plateau currents (normalized to the value measured in the absence of inhibitor) in the presence of 100 μM amiloride. Data are shown as mean ± SEM (each value represents an oocyte, n is shown in italic in each figure). Comparison between groups was performed by variance analysis (1-way ANOVA) followed by post hoc multiple comparison Tukey’s test. P

    Journal: JCI Insight

    Article Title: A variant of ASIC2 mediates sodium retention in nephrotic syndrome

    doi: 10.1172/jci.insight.148588

    Figure Lengend Snippet: Functional expression of ASIC2 in X. laevis oocyte. ( A ) ASIC2b coexpression attenuates ASIC2a desensitization. Original traces of whole-cell current (holding potential = –70 mV) in oocytes expressing ASIC2a alone or with ASIC2b, or t-ASIC2b, as indicated below the traces. Inward currents were induced by a rapid extracellular acidification from pH 7.4 to pH 4.0 (indicated by the horizontal bar). ( B ) Mean transient-induced (peak) and residual-induced (plateau) currents (corrected by currents recorded in control oocytes) in oocytes expressing ASIC2a alone (black bars) or coexpressing ASIC2b (red) or t-ASIC2b (blue). ( C ) Acid-induced plateau currents (normalized to maximal value achieved at pH 4) at different extracellular pH; colors as above. ( D ) Effect of amiloride: acid-induced (pH 4) plateau currents (normalized to the value measured in the absence of inhibitor) in the presence of 100 μM amiloride. Data are shown as mean ± SEM (each value represents an oocyte, n is shown in italic in each figure). Comparison between groups was performed by variance analysis (1-way ANOVA) followed by post hoc multiple comparison Tukey’s test. P

    Article Snippet: Samples were mixed with Laemmli buffer and proteins were separated by SDS-PAGE and analyzed by Western blot using pan-ASIC2 antibody as described above for CCDs.

    Techniques: Functional Assay, Expressing

    Expression of ASIC2 in nephrotic patients. ( A ) RT-qPCR analysis of ASIC2 mRNA in kidney biopsies from patients with NGN ( n = 11) or INS ( n = 8). Given the heterogeneity of biopsies in term of cell composition, data were standardized using distal nephron markers, as previously described ( 38 ). Data are in arbitrary units. ( B ) Immunolabeling of kidney serial sections with anti-AQP2 (red) and anti-ASIC2 antibody (b and d, green) from a patient with INS (upper panels) or a nonnephrotic patient (lower panels). Scale bar: 100 μm. Comparison between groups was performed by 2-tailed unpaired t test. P

    Journal: JCI Insight

    Article Title: A variant of ASIC2 mediates sodium retention in nephrotic syndrome

    doi: 10.1172/jci.insight.148588

    Figure Lengend Snippet: Expression of ASIC2 in nephrotic patients. ( A ) RT-qPCR analysis of ASIC2 mRNA in kidney biopsies from patients with NGN ( n = 11) or INS ( n = 8). Given the heterogeneity of biopsies in term of cell composition, data were standardized using distal nephron markers, as previously described ( 38 ). Data are in arbitrary units. ( B ) Immunolabeling of kidney serial sections with anti-AQP2 (red) and anti-ASIC2 antibody (b and d, green) from a patient with INS (upper panels) or a nonnephrotic patient (lower panels). Scale bar: 100 μm. Comparison between groups was performed by 2-tailed unpaired t test. P

    Article Snippet: Samples were mixed with Laemmli buffer and proteins were separated by SDS-PAGE and analyzed by Western blot using pan-ASIC2 antibody as described above for CCDs.

    Techniques: Expressing, Quantitative RT-PCR, Immunolabeling

    Long and truncated variants of ASIC2b. ( A ) 5′ terminal sequence of rat Asic2b cDNA (Accn1, transcript variant MDEG2, NM_012892, black) and of the short cDNA cloned from CC-PAN CCD (red, GenBank KP294334). The short sequence contains a putative translation initiation codon in frame with the ASIC2 ATG (in bold). ( B ) N-ter sequence of rat ASIC2b (black) and its putative truncated variant (red). The underlined sequence shows the first transmembrane domain. ( C ) Western blot analysis of ASIC2 expression in OKP and HEK cells transiently transfected with Asic2b or its truncated variant ( t-Asic2b ) or an empty vector. ASIC2b, acid-sensing ion channel 2b; CCDs, cortical-collecting ducts; CC, corticosteroid-clamped.

    Journal: JCI Insight

    Article Title: A variant of ASIC2 mediates sodium retention in nephrotic syndrome

    doi: 10.1172/jci.insight.148588

    Figure Lengend Snippet: Long and truncated variants of ASIC2b. ( A ) 5′ terminal sequence of rat Asic2b cDNA (Accn1, transcript variant MDEG2, NM_012892, black) and of the short cDNA cloned from CC-PAN CCD (red, GenBank KP294334). The short sequence contains a putative translation initiation codon in frame with the ASIC2 ATG (in bold). ( B ) N-ter sequence of rat ASIC2b (black) and its putative truncated variant (red). The underlined sequence shows the first transmembrane domain. ( C ) Western blot analysis of ASIC2 expression in OKP and HEK cells transiently transfected with Asic2b or its truncated variant ( t-Asic2b ) or an empty vector. ASIC2b, acid-sensing ion channel 2b; CCDs, cortical-collecting ducts; CC, corticosteroid-clamped.

    Article Snippet: Samples were mixed with Laemmli buffer and proteins were separated by SDS-PAGE and analyzed by Western blot using pan-ASIC2 antibody as described above for CCDs.

    Techniques: Sequencing, Variant Assay, Clone Assay, Western Blot, Expressing, Transfection, Plasmid Preparation

    Renal expression of ASIC2. ( A and B ) Western blot analysis of ASIC2 expression in kidney of WT and ASIC2b –/– CC-PAN rats and in CCDs of CC-Control and CC-PAN rats using a specific ASIC2a antibody ( A ) or a pan-ASIC2 antibody ( B ). Left, representative blot; right, densitometric analysis. Each value represents a rat. ( C ) Immunolabeling of isolated CCD from WT and ASIC2b –/– CC-Control and CC-PAN rats with a pan-ASIC2 antibody. Scale bar: 10 μm. ( D ) Immunolabelling of isolated CCD from PAN WT rat with a pan-ASIC2 antibody (green) and an anti-AE1 antibody (red). Comparison between groups was performed by 2-tailed unpaired t test. P

    Journal: JCI Insight

    Article Title: A variant of ASIC2 mediates sodium retention in nephrotic syndrome

    doi: 10.1172/jci.insight.148588

    Figure Lengend Snippet: Renal expression of ASIC2. ( A and B ) Western blot analysis of ASIC2 expression in kidney of WT and ASIC2b –/– CC-PAN rats and in CCDs of CC-Control and CC-PAN rats using a specific ASIC2a antibody ( A ) or a pan-ASIC2 antibody ( B ). Left, representative blot; right, densitometric analysis. Each value represents a rat. ( C ) Immunolabeling of isolated CCD from WT and ASIC2b –/– CC-Control and CC-PAN rats with a pan-ASIC2 antibody. Scale bar: 10 μm. ( D ) Immunolabelling of isolated CCD from PAN WT rat with a pan-ASIC2 antibody (green) and an anti-AE1 antibody (red). Comparison between groups was performed by 2-tailed unpaired t test. P

    Article Snippet: Samples were mixed with Laemmli buffer and proteins were separated by SDS-PAGE and analyzed by Western blot using pan-ASIC2 antibody as described above for CCDs.

    Techniques: Expressing, Western Blot, Immunolabeling, Isolation

    Glycosylation of t-ASIC2b. ( A ) Western blot analysis of ASIC2 expression in X . laevis oocytes injected with ASIC2b or t-ASIC2b cRNA or water and in protein extracts from CC-PAN rat kidneys. Samples were treated or not with N-glycosidase F (PNGase). ( B ) Original traces of whole-cell current (holding potential = –70 mV) in oocytes expressing ASIC2a with t-ASIC2b or a nonglycosylable form of truncated ASIC2b (ng-t-ASIC2b). Inward currents were induced by a rapid extracellular acidification from pH 7.4 to pH 4.0 (indicated by the horizontal bar). ( C ) Acid-induced (pH 4) plateau currents in the absence or presence of 100 μM amiloride. Data are shown as mean ± SEM (each value represents an oocyte, n is shown in italic in the figure). Comparison between groups was performed by 2-tailed unpaired t test. P

    Journal: JCI Insight

    Article Title: A variant of ASIC2 mediates sodium retention in nephrotic syndrome

    doi: 10.1172/jci.insight.148588

    Figure Lengend Snippet: Glycosylation of t-ASIC2b. ( A ) Western blot analysis of ASIC2 expression in X . laevis oocytes injected with ASIC2b or t-ASIC2b cRNA or water and in protein extracts from CC-PAN rat kidneys. Samples were treated or not with N-glycosidase F (PNGase). ( B ) Original traces of whole-cell current (holding potential = –70 mV) in oocytes expressing ASIC2a with t-ASIC2b or a nonglycosylable form of truncated ASIC2b (ng-t-ASIC2b). Inward currents were induced by a rapid extracellular acidification from pH 7.4 to pH 4.0 (indicated by the horizontal bar). ( C ) Acid-induced (pH 4) plateau currents in the absence or presence of 100 μM amiloride. Data are shown as mean ± SEM (each value represents an oocyte, n is shown in italic in the figure). Comparison between groups was performed by 2-tailed unpaired t test. P

    Article Snippet: Samples were mixed with Laemmli buffer and proteins were separated by SDS-PAGE and analyzed by Western blot using pan-ASIC2 antibody as described above for CCDs.

    Techniques: Western Blot, Expressing, Injection