c terminus rabbit anti aqp4 antibody  (Alomone Labs)


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    Alomone Labs c terminus rabbit anti aqp4 antibody
    Cytoprotective sera reduces binding of pathogenic <t>AQP4-IgG</t> and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).
    C Terminus Rabbit Anti Aqp4 Antibody, 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/c terminus rabbit anti aqp4 antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    c terminus rabbit anti aqp4 antibody - by Bioz Stars, 2022-05
    93/100 stars

    Images

    1) Product Images from "Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder"

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-01294-3

    Cytoprotective sera reduces binding of pathogenic AQP4-IgG and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).
    Figure Legend Snippet: Cytoprotective sera reduces binding of pathogenic AQP4-IgG and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).

    Techniques Used: Binding Assay, Expressing, Incubation, Staining

    Cytoprotection in a CDC assay conferred by a subset of AQP4-IgG seropositive NMOSD patient sera. ( A ) Cytoprotection assay in which CDC was measured by Alamar blue assay after incubation of AQP4-expressing cells with human complement (HC) and a mixture of cytotoxic AQP4-IgG (such as rAb-53) and heat-inactivated test serum. ( B ) CDC in AQP4-expressing CHO cells as a function of percentage of test NMOSD serum. CDC was produced by 0.5 μg/ml rAb-53 as cytotoxic AQP4-IgG (mean ± S.E.M., n = 4). Data shown for two control sera and four NMOSD patient sera that produced significant cytoprotection. ( C ) CDC measured as in B, but with cytotoxicity produced by monoclonal AQP4-IgGs rAb-10 or rAb-58 (each 5 μg/ml) (mean ± S.D., n = 4, * P
    Figure Legend Snippet: Cytoprotection in a CDC assay conferred by a subset of AQP4-IgG seropositive NMOSD patient sera. ( A ) Cytoprotection assay in which CDC was measured by Alamar blue assay after incubation of AQP4-expressing cells with human complement (HC) and a mixture of cytotoxic AQP4-IgG (such as rAb-53) and heat-inactivated test serum. ( B ) CDC in AQP4-expressing CHO cells as a function of percentage of test NMOSD serum. CDC was produced by 0.5 μg/ml rAb-53 as cytotoxic AQP4-IgG (mean ± S.E.M., n = 4). Data shown for two control sera and four NMOSD patient sera that produced significant cytoprotection. ( C ) CDC measured as in B, but with cytotoxicity produced by monoclonal AQP4-IgGs rAb-10 or rAb-58 (each 5 μg/ml) (mean ± S.D., n = 4, * P

    Techniques Used: CDC Assay, Alamar Blue Assay, Incubation, Expressing, Produced

    Potential mechanisms of cytoprotective IgG antibodies. Potential sites of action of cytoprotective IgGs include interference with complement activity, AQP4-IgG binding to cell-surface AQP4, supramolecular clustering of AQP4 or AQP4-IgG, and AQP4 cell-surface expression.
    Figure Legend Snippet: Potential mechanisms of cytoprotective IgG antibodies. Potential sites of action of cytoprotective IgGs include interference with complement activity, AQP4-IgG binding to cell-surface AQP4, supramolecular clustering of AQP4 or AQP4-IgG, and AQP4 cell-surface expression.

    Techniques Used: Activity Assay, Binding Assay, Expressing

    Cytoprotection is conferred by IgG antibodies and does not involve direct complement inhibition. ( A ) CDC measured in AQP4-expressing CHO cells, as in Fig. 2 A, but using purified IgG isolated from NMOSD or control sera (mean ± S.D., n = 4). ( B ) CDC measured as in Fig. 2 A, but using IgG-depleted cytoprotective NMOSD sera (mean ± S.D., n = 4, ** P
    Figure Legend Snippet: Cytoprotection is conferred by IgG antibodies and does not involve direct complement inhibition. ( A ) CDC measured in AQP4-expressing CHO cells, as in Fig. 2 A, but using purified IgG isolated from NMOSD or control sera (mean ± S.D., n = 4). ( B ) CDC measured as in Fig. 2 A, but using IgG-depleted cytoprotective NMOSD sera (mean ± S.D., n = 4, ** P

    Techniques Used: Inhibition, Expressing, Purification, Isolation

    Absence of AQP4-bound IgG2 and IgG4 subclass antibodies in cytoprotective sera. AQP4-expressing CHO cells incubated for 60 min with 10% control (no cytotoxicity) sera, cytoprotective sera, or high cytotoxicity sera. Bound antibody was revealed using fluorescent secondary antibodies selective for total IgG, IgG1, IgG2 and IgG4. Immunofluorescence micrographs representative of 2 sets of studies.
    Figure Legend Snippet: Absence of AQP4-bound IgG2 and IgG4 subclass antibodies in cytoprotective sera. AQP4-expressing CHO cells incubated for 60 min with 10% control (no cytotoxicity) sera, cytoprotective sera, or high cytotoxicity sera. Bound antibody was revealed using fluorescent secondary antibodies selective for total IgG, IgG1, IgG2 and IgG4. Immunofluorescence micrographs representative of 2 sets of studies.

    Techniques Used: Expressing, Incubation, Immunofluorescence

    Heterogeneity in complement-dependent cytotoxicity (CDC) produced by sera from AQP4-IgG seropositive NMOSD patients. ( A ) CDC assay, in which AQP4-expressing cells were incubated with human complement (HC) and AQP4-IgG (monoclonal antibody or heat-inactivated patient sera), with Alamar blue readout of cytotoxicity. ( B ) CDC in AQP4-expressing CHO cells produced by different concentrations of monoclonal AQP4-IgG rAb-53 (mean ± S.D., n = 4). ( C ) CDC produced by different percentages of NMOSD patient sera (mean ± S.D., n = 4). Curves for 7 different NMOSD patient sera shown. ( D ) (left) Scatter plot of CDC, expressed as the percentage of serum giving 50% killing, for 108 AQP4-IgG seropositive NMOSD sera and 25 (non-NMOSD) control sera. (Right) Histogram of CDC deduced from data on the left. ( E ) AQP4-IgG seropositivity study in which AQP4-IgG-expressing CHO cells were incubated with test sera, washed, and then immunostained for human IgG (red) and AQP4 (green). Micrographs shown for one control serum and three NMOSD sera which (in experiments as in C ) produced different levels (low, moderate, high) of CDC.
    Figure Legend Snippet: Heterogeneity in complement-dependent cytotoxicity (CDC) produced by sera from AQP4-IgG seropositive NMOSD patients. ( A ) CDC assay, in which AQP4-expressing cells were incubated with human complement (HC) and AQP4-IgG (monoclonal antibody or heat-inactivated patient sera), with Alamar blue readout of cytotoxicity. ( B ) CDC in AQP4-expressing CHO cells produced by different concentrations of monoclonal AQP4-IgG rAb-53 (mean ± S.D., n = 4). ( C ) CDC produced by different percentages of NMOSD patient sera (mean ± S.D., n = 4). Curves for 7 different NMOSD patient sera shown. ( D ) (left) Scatter plot of CDC, expressed as the percentage of serum giving 50% killing, for 108 AQP4-IgG seropositive NMOSD sera and 25 (non-NMOSD) control sera. (Right) Histogram of CDC deduced from data on the left. ( E ) AQP4-IgG seropositivity study in which AQP4-IgG-expressing CHO cells were incubated with test sera, washed, and then immunostained for human IgG (red) and AQP4 (green). Micrographs shown for one control serum and three NMOSD sera which (in experiments as in C ) produced different levels (low, moderate, high) of CDC.

    Techniques Used: Produced, CDC Assay, Expressing, Incubation

    Cytoprotective sera reduces apparent cell-surface AQP4 expression without affecting AQP4 clustering. ( A ) AQP4 immunofluorescence in AQP4-expressing CHO cells after 60 min incubation with 10% heat-inactivated control or cytoprotective NMOSD sera (or no treatment), followed by washing, fixation, cell permeabilization, and staining with C-terminal anti-AQP4 antibody and fluorescent secondary antibody (top). Total cellular fluorescence summarized below each fluorescence micrograph (mean ± S.D., n = 4, * P
    Figure Legend Snippet: Cytoprotective sera reduces apparent cell-surface AQP4 expression without affecting AQP4 clustering. ( A ) AQP4 immunofluorescence in AQP4-expressing CHO cells after 60 min incubation with 10% heat-inactivated control or cytoprotective NMOSD sera (or no treatment), followed by washing, fixation, cell permeabilization, and staining with C-terminal anti-AQP4 antibody and fluorescent secondary antibody (top). Total cellular fluorescence summarized below each fluorescence micrograph (mean ± S.D., n = 4, * P

    Techniques Used: Expressing, Immunofluorescence, Incubation, Staining, Fluorescence

    2) Product Images from "The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation"

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    Journal: Frontiers in Cellular Neuroscience

    doi: 10.3389/fncel.2017.00412

    Cav1 Y14 phosphorylation enhances AQP4 expression at the cell surface in MDA-435 cells. Immunoblot demonstrating the expression of the VSV-AQP4 transgene in MDA-435 cell lines carrying an empty vector, or expressing wild-type (WT), Y14F (dominant-negative) and Y14D (phosphomimetic) variants of Cav1, with β-actin shown as a loading control (A) . Cell-surface, intracellular, and total AQP4 levels in MDA-435 cells expressing WT, Y14F and Y14D Cav1 (B) . Quantification of the above ( C ; n = 3 independent experiments; * p
    Figure Legend Snippet: Cav1 Y14 phosphorylation enhances AQP4 expression at the cell surface in MDA-435 cells. Immunoblot demonstrating the expression of the VSV-AQP4 transgene in MDA-435 cell lines carrying an empty vector, or expressing wild-type (WT), Y14F (dominant-negative) and Y14D (phosphomimetic) variants of Cav1, with β-actin shown as a loading control (A) . Cell-surface, intracellular, and total AQP4 levels in MDA-435 cells expressing WT, Y14F and Y14D Cav1 (B) . Quantification of the above ( C ; n = 3 independent experiments; * p

    Techniques Used: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Dominant Negative Mutation

    H 2 O 2 increases AQP4 cell surface expression in a NAC-reversible manner. Immunoblots comparing cell-surface levels of AQP4 in astrocytes exposed to 200 and 400 μM H 2 O 2 for 1 h (A) or preincubated with NAC for 2 h prior to H 2 O 2 treatment (C) . Histograms depicting relative AQP4 cell-surface levels for the various conditions, normalized against the respective input levels for each condition ( B,D ; n = 3 independent experiments; * p
    Figure Legend Snippet: H 2 O 2 increases AQP4 cell surface expression in a NAC-reversible manner. Immunoblots comparing cell-surface levels of AQP4 in astrocytes exposed to 200 and 400 μM H 2 O 2 for 1 h (A) or preincubated with NAC for 2 h prior to H 2 O 2 treatment (C) . Histograms depicting relative AQP4 cell-surface levels for the various conditions, normalized against the respective input levels for each condition ( B,D ; n = 3 independent experiments; * p

    Techniques Used: Expressing, Western Blot

    The H 2 O 2 -induced increase in AQP4 cell surface expression is independent of AQP4 synthesis. Total AQP4 levels in cells incubated with 100 μg/mL cycloheximide for the indicated lengths of time with β-actin as a loading control (A) and densitometric quantification of the same ( B ; n = 3 independent experiments; * p
    Figure Legend Snippet: The H 2 O 2 -induced increase in AQP4 cell surface expression is independent of AQP4 synthesis. Total AQP4 levels in cells incubated with 100 μg/mL cycloheximide for the indicated lengths of time with β-actin as a loading control (A) and densitometric quantification of the same ( B ; n = 3 independent experiments; * p

    Techniques Used: Expressing, Incubation

    The Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppresses the H 2 O 2 -induced increase in AQP4 expression at the cell surface. Immunoblots comparing the level of caveolin-1 (Cav1) Y14 phosphorylation in astrocytes exposed to various concentrations of H 2 O 2 for 1 h (A) , or 200 μM H 2 O 2 for increasing lengths of time (B) . Immunoblot depicting the effects of 1 h pre-incubation with increasing concentrations of PP2 prior to an hour-long treatment with 200 μM H 2 O 2 on Cav1 Y14 phosphorylation (C) . Cell-surface, intracellular, and total AQP4 levels in control and PP2-pre-treated astrocytes in the presence and absence of a 1 h-long H 2 O 2 treatment (D) , and a histogram depicting the relative, input-normalized cell-surface AQP4 amounts under these various conditions ( E ; n = 3 independent experiments; * p
    Figure Legend Snippet: The Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppresses the H 2 O 2 -induced increase in AQP4 expression at the cell surface. Immunoblots comparing the level of caveolin-1 (Cav1) Y14 phosphorylation in astrocytes exposed to various concentrations of H 2 O 2 for 1 h (A) , or 200 μM H 2 O 2 for increasing lengths of time (B) . Immunoblot depicting the effects of 1 h pre-incubation with increasing concentrations of PP2 prior to an hour-long treatment with 200 μM H 2 O 2 on Cav1 Y14 phosphorylation (C) . Cell-surface, intracellular, and total AQP4 levels in control and PP2-pre-treated astrocytes in the presence and absence of a 1 h-long H 2 O 2 treatment (D) , and a histogram depicting the relative, input-normalized cell-surface AQP4 amounts under these various conditions ( E ; n = 3 independent experiments; * p

    Techniques Used: Expressing, Western Blot, Incubation

    Loss of Cav1 inhibits the H 2 O 2 -induced increase in AQP4 cell-surface expression in astrocytes. Cav1 expression in control- (siCtl) and Cav1-siRNA (siCav1)-transfected astrocytes (A) . Quantification of the above ( B ; n = 3 independent experiments; * p
    Figure Legend Snippet: Loss of Cav1 inhibits the H 2 O 2 -induced increase in AQP4 cell-surface expression in astrocytes. Cav1 expression in control- (siCtl) and Cav1-siRNA (siCav1)-transfected astrocytes (A) . Quantification of the above ( B ; n = 3 independent experiments; * p

    Techniques Used: Expressing, Transfection

    Hydrogen peroxide (H 2 O 2 ) increases aquaporin 4 (AQP4) protein expression levels in astrocytes and this effect is reversed by the antioxidant, N-acetylcysteine (NAC). Representative immunoblot of AQP4 in primary astrocyte cultures treated for 1 h with increasing concentrations of H 2 O 2 , with β-actin shown alongside as a loading control (A) . Densitometric analysis illustrating the relative differences in AQP4 levels (normalized against that of β-actin) for select concentrations of H 2 O 2 ( B ; averaged results of three independent experiments depicted). Immunoblot and quantification of AQP4 in astrocytes pre-incubated with the antioxidant NAC for 2 h, and then treated with H 2 O 2 for 1 h ( C,D ; n = 3 independent experiments quantified in D ). Arrows in immunoblots indicate the presence of multimers of AQP4. Statistically significant differences, as determined by the two-tailed Student’s t -test, are marked with symbols (* p
    Figure Legend Snippet: Hydrogen peroxide (H 2 O 2 ) increases aquaporin 4 (AQP4) protein expression levels in astrocytes and this effect is reversed by the antioxidant, N-acetylcysteine (NAC). Representative immunoblot of AQP4 in primary astrocyte cultures treated for 1 h with increasing concentrations of H 2 O 2 , with β-actin shown alongside as a loading control (A) . Densitometric analysis illustrating the relative differences in AQP4 levels (normalized against that of β-actin) for select concentrations of H 2 O 2 ( B ; averaged results of three independent experiments depicted). Immunoblot and quantification of AQP4 in astrocytes pre-incubated with the antioxidant NAC for 2 h, and then treated with H 2 O 2 for 1 h ( C,D ; n = 3 independent experiments quantified in D ). Arrows in immunoblots indicate the presence of multimers of AQP4. Statistically significant differences, as determined by the two-tailed Student’s t -test, are marked with symbols (* p

    Techniques Used: Expressing, Incubation, Western Blot, Two Tailed Test

    3) Product Images from "Vasopressin receptors V1a and V2 are not osmosensors"

    Article Title: Vasopressin receptors V1a and V2 are not osmosensors

    Journal: Physiological Reports

    doi: 10.14814/phy2.12519

    V1 a R-dependent downregulation of AQP4. (A) Volume traces obtained from an uninjected oocyte (left panel) and an AQP4/V1 a R-expressing oocyte challenged with an osmotic gradient of 50 mOsm mannitol for 30 sec. (B) Relative water permeability of oocytes expressing AQP4 (open circles; n = 5) or coexpressing AQP4/V1 a R (filled circles, n = 20) exposed to 1 μ mol/L vasopressin as marked by the black bar. (C) Relative water permeability of oocytes expressing AQP4 (open circles; n = 6) or coexpressing AQP4/V1 a R (filled circles; n = 22) when exposed to repeated osmotic challenges. (D) Relative water permeability of oocytes coexpressing AQP4/mGluR1a and exposed to 500 μ mol/L glutamate as indicated by the black bar (filled symbols, n = 8) or kept in control solution (open symbols, n = 8, not exposed to glutamate). The groups were compared with two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P
    Figure Legend Snippet: V1 a R-dependent downregulation of AQP4. (A) Volume traces obtained from an uninjected oocyte (left panel) and an AQP4/V1 a R-expressing oocyte challenged with an osmotic gradient of 50 mOsm mannitol for 30 sec. (B) Relative water permeability of oocytes expressing AQP4 (open circles; n = 5) or coexpressing AQP4/V1 a R (filled circles, n = 20) exposed to 1 μ mol/L vasopressin as marked by the black bar. (C) Relative water permeability of oocytes expressing AQP4 (open circles; n = 6) or coexpressing AQP4/V1 a R (filled circles; n = 22) when exposed to repeated osmotic challenges. (D) Relative water permeability of oocytes coexpressing AQP4/mGluR1a and exposed to 500 μ mol/L glutamate as indicated by the black bar (filled symbols, n = 8) or kept in control solution (open symbols, n = 8, not exposed to glutamate). The groups were compared with two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Techniques Used: Expressing, Permeability

    V1 a R-dependent internalization of AQP4. (A) Confocal laser scanning microscopy of oocytes expressing either AQP4 (left panel) or AQP4/V1 a R (right panel) immune-labeled for AQP4. The upper panels are representative images of oocytes exposed to control solution without vasopressin for 80 min. The middle panels are representative images of oocytes kept in control solution for 20 min and then treated with 1 μ mol/L vasopressin for 60 min. The lower panels are representative images of oocytes treated with a 50 mOsm hyperosmolar gradient for 30 sec every 10 min of an 80-min incubation period. (B) Oocyte plasma membrane fluorescence intensity normalized to that of the oocytes kept in control solution, n = 5 experiments with 3–6 oocytes per condition. The indicated groups were compared using one-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P
    Figure Legend Snippet: V1 a R-dependent internalization of AQP4. (A) Confocal laser scanning microscopy of oocytes expressing either AQP4 (left panel) or AQP4/V1 a R (right panel) immune-labeled for AQP4. The upper panels are representative images of oocytes exposed to control solution without vasopressin for 80 min. The middle panels are representative images of oocytes kept in control solution for 20 min and then treated with 1 μ mol/L vasopressin for 60 min. The lower panels are representative images of oocytes treated with a 50 mOsm hyperosmolar gradient for 30 sec every 10 min of an 80-min incubation period. (B) Oocyte plasma membrane fluorescence intensity normalized to that of the oocytes kept in control solution, n = 5 experiments with 3–6 oocytes per condition. The indicated groups were compared using one-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Techniques Used: Confocal Laser Scanning Microscopy, Expressing, Labeling, Incubation, Fluorescence

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    Alomone Labs c terminus rabbit anti aqp4 antibody
    Cytoprotective sera reduces binding of pathogenic <t>AQP4-IgG</t> and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).
    C Terminus Rabbit Anti Aqp4 Antibody, 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/c terminus rabbit anti aqp4 antibody/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    c terminus rabbit anti aqp4 antibody - by Bioz Stars, 2022-05
    93/100 stars
      Buy from Supplier

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    Cytoprotective sera reduces binding of pathogenic AQP4-IgG and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Cytoprotective sera reduces binding of pathogenic AQP4-IgG and C1q at the cell surface. ( A ) Cytoprotective serum binding study. AQP4-expressing CHO cells were incubated with 10% heat-inactivated cytoprotective or control sera (or 5 µg/ml rAb-53), followed by fluorescent anti-human IgG secondary antibody. ( B ) C1q and pathogenic AQP4-IgG (rAb-53) binding. AQP4-expressing CHO cells were incubated with heat-inactivated cytoprotective or control sera followed by 1 µg/ml rAb-53 and 10% C3-deficient human complement, and then stained for human IgG (red) and C1q (green).

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Binding Assay, Expressing, Incubation, Staining

    Cytoprotection in a CDC assay conferred by a subset of AQP4-IgG seropositive NMOSD patient sera. ( A ) Cytoprotection assay in which CDC was measured by Alamar blue assay after incubation of AQP4-expressing cells with human complement (HC) and a mixture of cytotoxic AQP4-IgG (such as rAb-53) and heat-inactivated test serum. ( B ) CDC in AQP4-expressing CHO cells as a function of percentage of test NMOSD serum. CDC was produced by 0.5 μg/ml rAb-53 as cytotoxic AQP4-IgG (mean ± S.E.M., n = 4). Data shown for two control sera and four NMOSD patient sera that produced significant cytoprotection. ( C ) CDC measured as in B, but with cytotoxicity produced by monoclonal AQP4-IgGs rAb-10 or rAb-58 (each 5 μg/ml) (mean ± S.D., n = 4, * P

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Cytoprotection in a CDC assay conferred by a subset of AQP4-IgG seropositive NMOSD patient sera. ( A ) Cytoprotection assay in which CDC was measured by Alamar blue assay after incubation of AQP4-expressing cells with human complement (HC) and a mixture of cytotoxic AQP4-IgG (such as rAb-53) and heat-inactivated test serum. ( B ) CDC in AQP4-expressing CHO cells as a function of percentage of test NMOSD serum. CDC was produced by 0.5 μg/ml rAb-53 as cytotoxic AQP4-IgG (mean ± S.E.M., n = 4). Data shown for two control sera and four NMOSD patient sera that produced significant cytoprotection. ( C ) CDC measured as in B, but with cytotoxicity produced by monoclonal AQP4-IgGs rAb-10 or rAb-58 (each 5 μg/ml) (mean ± S.D., n = 4, * P

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: CDC Assay, Alamar Blue Assay, Incubation, Expressing, Produced

    Potential mechanisms of cytoprotective IgG antibodies. Potential sites of action of cytoprotective IgGs include interference with complement activity, AQP4-IgG binding to cell-surface AQP4, supramolecular clustering of AQP4 or AQP4-IgG, and AQP4 cell-surface expression.

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Potential mechanisms of cytoprotective IgG antibodies. Potential sites of action of cytoprotective IgGs include interference with complement activity, AQP4-IgG binding to cell-surface AQP4, supramolecular clustering of AQP4 or AQP4-IgG, and AQP4 cell-surface expression.

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Activity Assay, Binding Assay, Expressing

    Cytoprotection is conferred by IgG antibodies and does not involve direct complement inhibition. ( A ) CDC measured in AQP4-expressing CHO cells, as in Fig. 2 A, but using purified IgG isolated from NMOSD or control sera (mean ± S.D., n = 4). ( B ) CDC measured as in Fig. 2 A, but using IgG-depleted cytoprotective NMOSD sera (mean ± S.D., n = 4, ** P

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Cytoprotection is conferred by IgG antibodies and does not involve direct complement inhibition. ( A ) CDC measured in AQP4-expressing CHO cells, as in Fig. 2 A, but using purified IgG isolated from NMOSD or control sera (mean ± S.D., n = 4). ( B ) CDC measured as in Fig. 2 A, but using IgG-depleted cytoprotective NMOSD sera (mean ± S.D., n = 4, ** P

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Inhibition, Expressing, Purification, Isolation

    Absence of AQP4-bound IgG2 and IgG4 subclass antibodies in cytoprotective sera. AQP4-expressing CHO cells incubated for 60 min with 10% control (no cytotoxicity) sera, cytoprotective sera, or high cytotoxicity sera. Bound antibody was revealed using fluorescent secondary antibodies selective for total IgG, IgG1, IgG2 and IgG4. Immunofluorescence micrographs representative of 2 sets of studies.

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Absence of AQP4-bound IgG2 and IgG4 subclass antibodies in cytoprotective sera. AQP4-expressing CHO cells incubated for 60 min with 10% control (no cytotoxicity) sera, cytoprotective sera, or high cytotoxicity sera. Bound antibody was revealed using fluorescent secondary antibodies selective for total IgG, IgG1, IgG2 and IgG4. Immunofluorescence micrographs representative of 2 sets of studies.

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Expressing, Incubation, Immunofluorescence

    Heterogeneity in complement-dependent cytotoxicity (CDC) produced by sera from AQP4-IgG seropositive NMOSD patients. ( A ) CDC assay, in which AQP4-expressing cells were incubated with human complement (HC) and AQP4-IgG (monoclonal antibody or heat-inactivated patient sera), with Alamar blue readout of cytotoxicity. ( B ) CDC in AQP4-expressing CHO cells produced by different concentrations of monoclonal AQP4-IgG rAb-53 (mean ± S.D., n = 4). ( C ) CDC produced by different percentages of NMOSD patient sera (mean ± S.D., n = 4). Curves for 7 different NMOSD patient sera shown. ( D ) (left) Scatter plot of CDC, expressed as the percentage of serum giving 50% killing, for 108 AQP4-IgG seropositive NMOSD sera and 25 (non-NMOSD) control sera. (Right) Histogram of CDC deduced from data on the left. ( E ) AQP4-IgG seropositivity study in which AQP4-IgG-expressing CHO cells were incubated with test sera, washed, and then immunostained for human IgG (red) and AQP4 (green). Micrographs shown for one control serum and three NMOSD sera which (in experiments as in C ) produced different levels (low, moderate, high) of CDC.

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Heterogeneity in complement-dependent cytotoxicity (CDC) produced by sera from AQP4-IgG seropositive NMOSD patients. ( A ) CDC assay, in which AQP4-expressing cells were incubated with human complement (HC) and AQP4-IgG (monoclonal antibody or heat-inactivated patient sera), with Alamar blue readout of cytotoxicity. ( B ) CDC in AQP4-expressing CHO cells produced by different concentrations of monoclonal AQP4-IgG rAb-53 (mean ± S.D., n = 4). ( C ) CDC produced by different percentages of NMOSD patient sera (mean ± S.D., n = 4). Curves for 7 different NMOSD patient sera shown. ( D ) (left) Scatter plot of CDC, expressed as the percentage of serum giving 50% killing, for 108 AQP4-IgG seropositive NMOSD sera and 25 (non-NMOSD) control sera. (Right) Histogram of CDC deduced from data on the left. ( E ) AQP4-IgG seropositivity study in which AQP4-IgG-expressing CHO cells were incubated with test sera, washed, and then immunostained for human IgG (red) and AQP4 (green). Micrographs shown for one control serum and three NMOSD sera which (in experiments as in C ) produced different levels (low, moderate, high) of CDC.

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Produced, CDC Assay, Expressing, Incubation

    Cytoprotective sera reduces apparent cell-surface AQP4 expression without affecting AQP4 clustering. ( A ) AQP4 immunofluorescence in AQP4-expressing CHO cells after 60 min incubation with 10% heat-inactivated control or cytoprotective NMOSD sera (or no treatment), followed by washing, fixation, cell permeabilization, and staining with C-terminal anti-AQP4 antibody and fluorescent secondary antibody (top). Total cellular fluorescence summarized below each fluorescence micrograph (mean ± S.D., n = 4, * P

    Journal: Scientific Reports

    Article Title: Cytoprotective IgG antibodies in sera from a subset of patients with AQP4-IgG seropositive neuromyelitis optica spectrum disorder

    doi: 10.1038/s41598-021-01294-3

    Figure Lengend Snippet: Cytoprotective sera reduces apparent cell-surface AQP4 expression without affecting AQP4 clustering. ( A ) AQP4 immunofluorescence in AQP4-expressing CHO cells after 60 min incubation with 10% heat-inactivated control or cytoprotective NMOSD sera (or no treatment), followed by washing, fixation, cell permeabilization, and staining with C-terminal anti-AQP4 antibody and fluorescent secondary antibody (top). Total cellular fluorescence summarized below each fluorescence micrograph (mean ± S.D., n = 4, * P

    Article Snippet: To study the effect of patient sera on AQP4 expression, CHO-AQP4 cells were treated with 10% heat-inactivated patient sera for 1 h, and cells were then fixed in 4% PFA for 15 min and permeabilized with 0.1% Triton-X for 10 min. After blocking again with blocking buffer, cells were incubated with a C-terminus rabbit anti-AQP4 antibody (anti-AQP4 249-323 antibody, 1:1,000, Alomone Labs, Jerusalem, Israel) for 1 h, followed by goat anti-rabbit IgG-conjugated Alexa Fluor-488 secondary antibody (1:500, Invitrogen).

    Techniques: Expressing, Immunofluorescence, Incubation, Staining, Fluorescence

    Cav1 Y14 phosphorylation enhances AQP4 expression at the cell surface in MDA-435 cells. Immunoblot demonstrating the expression of the VSV-AQP4 transgene in MDA-435 cell lines carrying an empty vector, or expressing wild-type (WT), Y14F (dominant-negative) and Y14D (phosphomimetic) variants of Cav1, with β-actin shown as a loading control (A) . Cell-surface, intracellular, and total AQP4 levels in MDA-435 cells expressing WT, Y14F and Y14D Cav1 (B) . Quantification of the above ( C ; n = 3 independent experiments; * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: Cav1 Y14 phosphorylation enhances AQP4 expression at the cell surface in MDA-435 cells. Immunoblot demonstrating the expression of the VSV-AQP4 transgene in MDA-435 cell lines carrying an empty vector, or expressing wild-type (WT), Y14F (dominant-negative) and Y14D (phosphomimetic) variants of Cav1, with β-actin shown as a loading control (A) . Cell-surface, intracellular, and total AQP4 levels in MDA-435 cells expressing WT, Y14F and Y14D Cav1 (B) . Quantification of the above ( C ; n = 3 independent experiments; * p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Multiple Displacement Amplification, Plasmid Preparation, Dominant Negative Mutation

    H 2 O 2 increases AQP4 cell surface expression in a NAC-reversible manner. Immunoblots comparing cell-surface levels of AQP4 in astrocytes exposed to 200 and 400 μM H 2 O 2 for 1 h (A) or preincubated with NAC for 2 h prior to H 2 O 2 treatment (C) . Histograms depicting relative AQP4 cell-surface levels for the various conditions, normalized against the respective input levels for each condition ( B,D ; n = 3 independent experiments; * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: H 2 O 2 increases AQP4 cell surface expression in a NAC-reversible manner. Immunoblots comparing cell-surface levels of AQP4 in astrocytes exposed to 200 and 400 μM H 2 O 2 for 1 h (A) or preincubated with NAC for 2 h prior to H 2 O 2 treatment (C) . Histograms depicting relative AQP4 cell-surface levels for the various conditions, normalized against the respective input levels for each condition ( B,D ; n = 3 independent experiments; * p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Western Blot

    The H 2 O 2 -induced increase in AQP4 cell surface expression is independent of AQP4 synthesis. Total AQP4 levels in cells incubated with 100 μg/mL cycloheximide for the indicated lengths of time with β-actin as a loading control (A) and densitometric quantification of the same ( B ; n = 3 independent experiments; * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: The H 2 O 2 -induced increase in AQP4 cell surface expression is independent of AQP4 synthesis. Total AQP4 levels in cells incubated with 100 μg/mL cycloheximide for the indicated lengths of time with β-actin as a loading control (A) and densitometric quantification of the same ( B ; n = 3 independent experiments; * p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Incubation

    The Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppresses the H 2 O 2 -induced increase in AQP4 expression at the cell surface. Immunoblots comparing the level of caveolin-1 (Cav1) Y14 phosphorylation in astrocytes exposed to various concentrations of H 2 O 2 for 1 h (A) , or 200 μM H 2 O 2 for increasing lengths of time (B) . Immunoblot depicting the effects of 1 h pre-incubation with increasing concentrations of PP2 prior to an hour-long treatment with 200 μM H 2 O 2 on Cav1 Y14 phosphorylation (C) . Cell-surface, intracellular, and total AQP4 levels in control and PP2-pre-treated astrocytes in the presence and absence of a 1 h-long H 2 O 2 treatment (D) , and a histogram depicting the relative, input-normalized cell-surface AQP4 amounts under these various conditions ( E ; n = 3 independent experiments; * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: The Src kinase inhibitor 4-Amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) suppresses the H 2 O 2 -induced increase in AQP4 expression at the cell surface. Immunoblots comparing the level of caveolin-1 (Cav1) Y14 phosphorylation in astrocytes exposed to various concentrations of H 2 O 2 for 1 h (A) , or 200 μM H 2 O 2 for increasing lengths of time (B) . Immunoblot depicting the effects of 1 h pre-incubation with increasing concentrations of PP2 prior to an hour-long treatment with 200 μM H 2 O 2 on Cav1 Y14 phosphorylation (C) . Cell-surface, intracellular, and total AQP4 levels in control and PP2-pre-treated astrocytes in the presence and absence of a 1 h-long H 2 O 2 treatment (D) , and a histogram depicting the relative, input-normalized cell-surface AQP4 amounts under these various conditions ( E ; n = 3 independent experiments; * p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Western Blot, Incubation

    Loss of Cav1 inhibits the H 2 O 2 -induced increase in AQP4 cell-surface expression in astrocytes. Cav1 expression in control- (siCtl) and Cav1-siRNA (siCav1)-transfected astrocytes (A) . Quantification of the above ( B ; n = 3 independent experiments; * p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: Loss of Cav1 inhibits the H 2 O 2 -induced increase in AQP4 cell-surface expression in astrocytes. Cav1 expression in control- (siCtl) and Cav1-siRNA (siCav1)-transfected astrocytes (A) . Quantification of the above ( B ; n = 3 independent experiments; * p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Transfection

    Hydrogen peroxide (H 2 O 2 ) increases aquaporin 4 (AQP4) protein expression levels in astrocytes and this effect is reversed by the antioxidant, N-acetylcysteine (NAC). Representative immunoblot of AQP4 in primary astrocyte cultures treated for 1 h with increasing concentrations of H 2 O 2 , with β-actin shown alongside as a loading control (A) . Densitometric analysis illustrating the relative differences in AQP4 levels (normalized against that of β-actin) for select concentrations of H 2 O 2 ( B ; averaged results of three independent experiments depicted). Immunoblot and quantification of AQP4 in astrocytes pre-incubated with the antioxidant NAC for 2 h, and then treated with H 2 O 2 for 1 h ( C,D ; n = 3 independent experiments quantified in D ). Arrows in immunoblots indicate the presence of multimers of AQP4. Statistically significant differences, as determined by the two-tailed Student’s t -test, are marked with symbols (* p

    Journal: Frontiers in Cellular Neuroscience

    Article Title: The Oxidative Stress-Induced Increase in the Membrane Expression of the Water-Permeable Channel Aquaporin-4 in Astrocytes Is Regulated by Caveolin-1 Phosphorylation

    doi: 10.3389/fncel.2017.00412

    Figure Lengend Snippet: Hydrogen peroxide (H 2 O 2 ) increases aquaporin 4 (AQP4) protein expression levels in astrocytes and this effect is reversed by the antioxidant, N-acetylcysteine (NAC). Representative immunoblot of AQP4 in primary astrocyte cultures treated for 1 h with increasing concentrations of H 2 O 2 , with β-actin shown alongside as a loading control (A) . Densitometric analysis illustrating the relative differences in AQP4 levels (normalized against that of β-actin) for select concentrations of H 2 O 2 ( B ; averaged results of three independent experiments depicted). Immunoblot and quantification of AQP4 in astrocytes pre-incubated with the antioxidant NAC for 2 h, and then treated with H 2 O 2 for 1 h ( C,D ; n = 3 independent experiments quantified in D ). Arrows in immunoblots indicate the presence of multimers of AQP4. Statistically significant differences, as determined by the two-tailed Student’s t -test, are marked with symbols (* p

    Article Snippet: Antibodies and Reagents The following antibodies were used in the present study: rabbit polyclonal anti-AQP4 targeting residues 249–323 of rat AQP4 ( RRID:AB_2039734 ; catalog no. AQP-004, lot no. AQP004AN1302; Alomone Laboratories, Jerusalem, Israel), rabbit polyclonal anti-Cav1 raised against the N-terminus of the human sequence ( RRID:AB_2072042 ; catalog no. sc-894, lot no. H0307; Santa Cruz Biotechnology, Santa Cruz, CA, USA), mouse monoclonal anti-Cav1-Y14 ( RRID:AB_2244199 catalog no. 3251S, lot no. 2; Cell Signaling Technology, USA) and mouse monoclonal anti-β-actin ( RRID:AB_476744; catalog no. A5441, lot no. 064K4790; Sigma-Aldrich, St.Louis, MO, USA).

    Techniques: Expressing, Incubation, Western Blot, Two Tailed Test

    V1 a R-dependent downregulation of AQP4. (A) Volume traces obtained from an uninjected oocyte (left panel) and an AQP4/V1 a R-expressing oocyte challenged with an osmotic gradient of 50 mOsm mannitol for 30 sec. (B) Relative water permeability of oocytes expressing AQP4 (open circles; n = 5) or coexpressing AQP4/V1 a R (filled circles, n = 20) exposed to 1 μ mol/L vasopressin as marked by the black bar. (C) Relative water permeability of oocytes expressing AQP4 (open circles; n = 6) or coexpressing AQP4/V1 a R (filled circles; n = 22) when exposed to repeated osmotic challenges. (D) Relative water permeability of oocytes coexpressing AQP4/mGluR1a and exposed to 500 μ mol/L glutamate as indicated by the black bar (filled symbols, n = 8) or kept in control solution (open symbols, n = 8, not exposed to glutamate). The groups were compared with two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Journal: Physiological Reports

    Article Title: Vasopressin receptors V1a and V2 are not osmosensors

    doi: 10.14814/phy2.12519

    Figure Lengend Snippet: V1 a R-dependent downregulation of AQP4. (A) Volume traces obtained from an uninjected oocyte (left panel) and an AQP4/V1 a R-expressing oocyte challenged with an osmotic gradient of 50 mOsm mannitol for 30 sec. (B) Relative water permeability of oocytes expressing AQP4 (open circles; n = 5) or coexpressing AQP4/V1 a R (filled circles, n = 20) exposed to 1 μ mol/L vasopressin as marked by the black bar. (C) Relative water permeability of oocytes expressing AQP4 (open circles; n = 6) or coexpressing AQP4/V1 a R (filled circles; n = 22) when exposed to repeated osmotic challenges. (D) Relative water permeability of oocytes coexpressing AQP4/mGluR1a and exposed to 500 μ mol/L glutamate as indicated by the black bar (filled symbols, n = 8) or kept in control solution (open symbols, n = 8, not exposed to glutamate). The groups were compared with two-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Article Snippet: Sections of 2 μm were cut on a Leica RM 2126 microtome and immunostaining performed as described previously (Fenton et al. ) using a rabbit polyclonal anti-AQP4 antibody 1:5000 (Alamone Laboratories, Jerusalem, Israel).

    Techniques: Expressing, Permeability

    V1 a R-dependent internalization of AQP4. (A) Confocal laser scanning microscopy of oocytes expressing either AQP4 (left panel) or AQP4/V1 a R (right panel) immune-labeled for AQP4. The upper panels are representative images of oocytes exposed to control solution without vasopressin for 80 min. The middle panels are representative images of oocytes kept in control solution for 20 min and then treated with 1 μ mol/L vasopressin for 60 min. The lower panels are representative images of oocytes treated with a 50 mOsm hyperosmolar gradient for 30 sec every 10 min of an 80-min incubation period. (B) Oocyte plasma membrane fluorescence intensity normalized to that of the oocytes kept in control solution, n = 5 experiments with 3–6 oocytes per condition. The indicated groups were compared using one-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Journal: Physiological Reports

    Article Title: Vasopressin receptors V1a and V2 are not osmosensors

    doi: 10.14814/phy2.12519

    Figure Lengend Snippet: V1 a R-dependent internalization of AQP4. (A) Confocal laser scanning microscopy of oocytes expressing either AQP4 (left panel) or AQP4/V1 a R (right panel) immune-labeled for AQP4. The upper panels are representative images of oocytes exposed to control solution without vasopressin for 80 min. The middle panels are representative images of oocytes kept in control solution for 20 min and then treated with 1 μ mol/L vasopressin for 60 min. The lower panels are representative images of oocytes treated with a 50 mOsm hyperosmolar gradient for 30 sec every 10 min of an 80-min incubation period. (B) Oocyte plasma membrane fluorescence intensity normalized to that of the oocytes kept in control solution, n = 5 experiments with 3–6 oocytes per condition. The indicated groups were compared using one-way analysis of variance (ANOVA) with Šídák’s multiple comparison post hoc test. * P

    Article Snippet: Sections of 2 μm were cut on a Leica RM 2126 microtome and immunostaining performed as described previously (Fenton et al. ) using a rabbit polyclonal anti-AQP4 antibody 1:5000 (Alamone Laboratories, Jerusalem, Israel).

    Techniques: Confocal Laser Scanning Microscopy, Expressing, Labeling, Incubation, Fluorescence