asic1a  (Alomone Labs)

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

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

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

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

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

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

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

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

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

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