ly294002  (Alomone Labs)


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    Name:
    LY 294002
    Description:
    LY 294002 is a potent and specific cell permeable inhibitor of Pl 3 kinase
    Catalog Number:
    L-300
    Price:
    62.0
    Category:
    Small Molecule
    Source:
    Synthetic
    Applications:
    0
    Purity:
    >99%
    Size:
    1 mg
    Format:
    Lyophilized/solid.
    Formula:
    C19H17NO3
    Molecular Weight:
    307.35
    Molecule Name:
    2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride.
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    Structured Review

    Alomone Labs ly294002
    LY 294002
    LY 294002 is a potent and specific cell permeable inhibitor of Pl 3 kinase
    https://www.bioz.com/result/ly294002/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ly294002 - by Bioz Stars, 2021-09
    94/100 stars

    Images

    1) Product Images from "Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *"

    Article Title: Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.033480

    Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior
    Figure Legend Snippet: Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior

    Techniques Used: Incubation

    2) Product Images from "Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *"

    Article Title: Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.033480

    Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior
    Figure Legend Snippet: Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior

    Techniques Used: Incubation

    3) Product Images from "PI3K?-Dependent Signaling in Mouse Olfactory Receptor Neurons"

    Article Title: PI3K?-Dependent Signaling in Mouse Olfactory Receptor Neurons

    Journal: Chemical Senses

    doi: 10.1093/chemse/bjq020

    PI3Kγ KO mice show a large reduction in PI3K-mediated signaling upon odorant stimulation. (A) PI3Kγ KO mice show effects of odorant response enhancement to the pan-specific PI3K inhibitors wortmannin and LY294002 in a reduced number of
    Figure Legend Snippet: PI3Kγ KO mice show a large reduction in PI3K-mediated signaling upon odorant stimulation. (A) PI3Kγ KO mice show effects of odorant response enhancement to the pan-specific PI3K inhibitors wortmannin and LY294002 in a reduced number of

    Techniques Used: Mouse Assay

    Odorant-induced PI3K-dependent signaling in mouse ORNs. (A) ORNs from wt mice show enhanced responses to complex odorant stimulation following inhibition of PI3K by wortmannin and LY294002. Cells for analysis were selected by responsiveness to H100 in
    Figure Legend Snippet: Odorant-induced PI3K-dependent signaling in mouse ORNs. (A) ORNs from wt mice show enhanced responses to complex odorant stimulation following inhibition of PI3K by wortmannin and LY294002. Cells for analysis were selected by responsiveness to H100 in

    Techniques Used: Mouse Assay, Inhibition

    4) Product Images from "Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *"

    Article Title: Hypoxia Up-regulates CD36 Expression and Function via Hypoxia-inducible Factor-1- and Phosphatidylinositol 3-Kinase-dependent Mechanisms *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.033480

    Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior
    Figure Legend Snippet: Implication of the PI3K pathway in the hypoxic accumulation of CD36. ARPEs were incubated with or without PI3K ( A ) (wortmannin, 200 n m ; LY294002, 20 μ m ) and mTOR (rapamycin, 100 n m ) inhibitors, or PI3K p85α siRNA ( B and C ) (30 n m ) prior

    Techniques Used: Incubation

    5) Product Images from "Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2 *"

    Article Title: Insulin Growth Factor-2 Binding Protein 3 (IGF2BP3) Is a Glioblastoma-specific Marker That Activates Phosphatidylinositol 3-Kinase/Mitogen-activated Protein Kinase (PI3K/MAPK) Pathways by Modulating IGF-2 *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.178012

    IMP3 exerts its proproliferative effects through the IGF-2-PI3K and IGF-2-MAPK cascades. A , viability was measured by MTT assay at indicated time points for H1299 vector and IMP3 stable clones treated with a neutralizing antibody for IGF-2 as indicated. The assays were carried out in triplicate, and the mean value for each cell type at each time point was used to generate the graph. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups and a p ≤ 0.05 is represented with an asterisk , p ≤ 0.01 is represented as double asterisks and p ≤ 0.0001 is represented as triple asterisks. B , viability was measured by MTT assay as an indicator of cell proliferation at the indicated time points for either mock, cyclophilin ( cyclo. ), or IMP3 siRNA-transfected U138 cells. IGF-2 was exogenously supplemented as indicated to monitor its ability to rescue the effects of IMP3 knockdown. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups. A p value ≤ 0.05 is represented as an asterisk , which is significant, and NS refers to nonsignificant difference. C , equal amounts of total protein lysates from cyclophilin and IMP3 siRNA-treated U138 cells were subjected to Western blotting with the indicated antibodies. Note that the intensity of phospho-mTOR, phospho-Akt, phospho-4EBP1, phospho-MEK1/2, and phospho-ERK1/2 bands reduces in lane 2 compared with lane 1 , whereas that of total the corresponding total proteins remain unchanged. D , viability was measured by MTT assay on day 9 after plating for the U373 vector clone (#6) and IMP3 stable clones (#5 and 17) treated with dimethyl sulfoxide (vehicle), LY294002 (20 μ m ), or U0126 (10 μ m ). Please note that dimethyl sulfoxide ( DMSO )-treated IMP3 stable clone (#5 and #17) grow faster than vector stable clone (compare light gray bars ). Both LY294002 and U0126 treated IMP3 stable clone (#5 and #17) failed to show faster growth than vector stable clone (compare dark gray and black bars with light gray bars ).
    Figure Legend Snippet: IMP3 exerts its proproliferative effects through the IGF-2-PI3K and IGF-2-MAPK cascades. A , viability was measured by MTT assay at indicated time points for H1299 vector and IMP3 stable clones treated with a neutralizing antibody for IGF-2 as indicated. The assays were carried out in triplicate, and the mean value for each cell type at each time point was used to generate the graph. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups and a p ≤ 0.05 is represented with an asterisk , p ≤ 0.01 is represented as double asterisks and p ≤ 0.0001 is represented as triple asterisks. B , viability was measured by MTT assay as an indicator of cell proliferation at the indicated time points for either mock, cyclophilin ( cyclo. ), or IMP3 siRNA-transfected U138 cells. IGF-2 was exogenously supplemented as indicated to monitor its ability to rescue the effects of IMP3 knockdown. A one-way analysis of variance was carried out to test the significance of the observed differences between the groups. A p value ≤ 0.05 is represented as an asterisk , which is significant, and NS refers to nonsignificant difference. C , equal amounts of total protein lysates from cyclophilin and IMP3 siRNA-treated U138 cells were subjected to Western blotting with the indicated antibodies. Note that the intensity of phospho-mTOR, phospho-Akt, phospho-4EBP1, phospho-MEK1/2, and phospho-ERK1/2 bands reduces in lane 2 compared with lane 1 , whereas that of total the corresponding total proteins remain unchanged. D , viability was measured by MTT assay on day 9 after plating for the U373 vector clone (#6) and IMP3 stable clones (#5 and 17) treated with dimethyl sulfoxide (vehicle), LY294002 (20 μ m ), or U0126 (10 μ m ). Please note that dimethyl sulfoxide ( DMSO )-treated IMP3 stable clone (#5 and #17) grow faster than vector stable clone (compare light gray bars ). Both LY294002 and U0126 treated IMP3 stable clone (#5 and #17) failed to show faster growth than vector stable clone (compare dark gray and black bars with light gray bars ).

    Techniques Used: MTT Assay, Plasmid Preparation, Clone Assay, Transfection, Western Blot, Stable Transfection

    6) Product Images from "Fatty acid synthase as a novel target for meningioma therapy"

    Article Title: Fatty acid synthase as a novel target for meningioma therapy

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/noq004

    FAS expression can be induced in serum-starved IOMM-Lee cells by insulin stimulation (A). Inhibition of PI3K signaling by wortmannin or Ly294002 effectively downregulates the FAS levels in serum-starved IOMM-Lee cells stimulated with insulin, while inhibition
    Figure Legend Snippet: FAS expression can be induced in serum-starved IOMM-Lee cells by insulin stimulation (A). Inhibition of PI3K signaling by wortmannin or Ly294002 effectively downregulates the FAS levels in serum-starved IOMM-Lee cells stimulated with insulin, while inhibition

    Techniques Used: Expressing, Inhibition

    Related Articles

    other:

    Article Title: PML-Dependent Memory of Type I Interferon Treatment Results in a Restricted Form of HSV Latency
    Article Snippet: ReagentsCompounds used in the study are as follows: Acycloguanosine, FUDR, LY 294002, Nerve Growth Factor 2.5S (Alomone Labs), Primocin (Invivogen), Aphidicolin (AG Scientific), IFN-α (EMD Millipore IF009), IFN-β (EMD Millipore IF011), IFN-γ (EMD Millipore IF005), IFN-λ2 (PeproTech 250-33); WAY-150138 was kindly provided by Pfizer, Dr. Jay Brown and Dr. Dan Engel at the University of Virginia, and Dr. Lynn Enquist at Princeton University.

    Article Title: PI3K?-Dependent Signaling in Mouse Olfactory Receptor Neurons
    Article Snippet: Forskolin and 3-isobutyl-1-methylxanthine (IBMX) were obtained from Sigma-Aldrich, Wortmannin and LY294002 from Alomone Labs, and AS252424 and TGX-221 from Cayman Chemicals.

    Mouse Assay:

    Article Title: PI3K?-Dependent Signaling in Mouse Olfactory Receptor Neurons
    Article Snippet: .. Only 11.8 ± 3% of the cells per animal from the PI3Kγ KO mice capable of showing an enhanced response ( n = 50 cells from 3 animals) were sensitive to wortmannin treatment and only 9.5 ± 2.8% of the cells were sensitive to LY294002, compared with 36.9 ± 4.5% and 32.4 ± 5.2%, respectively, of the cells from wt mice. ..

    Article Title: PI3K?-Dependent Signaling in Mouse Olfactory Receptor Neurons
    Article Snippet: .. However, in contrast to ORNs from wt mice, the pan-specific PI3K inhibitors wortmannin and LY294002 had a significantly lower incidence of effect on ORNs from the PI3Kγ KO mice compared with those from wt mice ( ). ..

    Binding Assay:

    Article Title: The TLR9 agonist, CpG-ODN 1826, ameliorates cardiac dysfunction after trauma-hemorrhage
    Article Snippet: .. LY294002 is considered as a specifically competitive inhibitor for the ATP binding site of phosphatidylinositol 3-kinase (PI3K) and was purchased from Alomone labs Ltd, Israel. ..

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    Alomone Labs rabbit α hcn2 antibody
    Domains mediating the interaction of VAPB with <t>HCN2.</t> A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P
    Rabbit α Hcn2 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit α hcn2 antibody/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
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    Alomone Labs rabbit anti kcnj10
    <t>Kcnj10</t> and Kcnq1 are expressed in the stria vascularis of Atp6v0a4 mutant mice. (A,C) Mid-modiolar sections of the inner ear of control (A) and mutant (C) mice at P20. Regions in boxes in A and C are magnified in B and D, respectively. In wild types and mutants, a similar Kcnj10 expression pattern is observed. Especially strong expression is found in the intermediate cells of the stria vascularis (B,D, black arrowheads), whereas the marginal cells are negative (B,D, red arrowheads). It is also noticeable that mutants examined ( n =5) did not seem to show Reissner's membrane so much distended towards the scala vestibuli as at P0 and P5. We think this might be due to the decalcification process used to facilitate the sectioning of these samples, or to the resolution of any pressure difference across Reissner's membrane by rupture. (E,F) Expression of Kcnq1 is specifically found in the marginal cells of the stria vascularis at P20 in both wild types and mutants (E,F, red arrowheads). RM, Reissner's membrane; SV, stria vascularis. Scale bars: 100 μm (A,C); 20 μm (B,D-F).
    Rabbit Anti Kcnj10, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs anti aqp4
    The synthesized GILZ-p inhibited LPS induced Müller cell gliosis. Western blot analysis was performed to determine the protein expression levels of glial fibrillary acidic protein (GFAP) (A,B) , and <t>AQP4</t> (C,D) in Müller cells treated with 1000 ng/ml LPS in combination with different concentrations of GILZ-p (0.01, 0.1, 1, and 10 μM) for 24 h. β-actin was used as the loading control. The results of quantitative analysis, as determined by densitometric analysis, were expressed as relative to β-actin. Data represent the mean ± SE; the Mann–Whitney U -test was used for comparisons between two groups. n = 3 for each group. ∗ P
    Anti Aqp4, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Domains mediating the interaction of VAPB with HCN2. A ) Schematic illustration of a HCN subunit. The CNBD and some of the truncation constructs studied are indicated. B ) All truncation constructs exhibited a positive interaction, evident from growth on -LWHA dropout medium. C ) Representative current traces and the relative currents for different C-terminal deletions expressed alone or with VAPB. D ) Representative current traces and the relative current amplitudes for the N-terminal truncated NTK HCN2 expressed alone or with VAPB. E ) Relative current amplitudes of NTK HCN2 HA Ex (extracellular HA-tag) expressed alone or with VAPB. F ) Relative surface expression of NTK HCN2 HA Ex expressed alone or with VAPB analyzed as relative light units (RLUs). G ) Schematic illustration, representative traces, and currents of a HCN2 channel chimera with the N terminus of HCN4 ( HCN4-N HCN2) expressed alone or with VAPB. H ) Relative currents of HCN2 expressed alone or coexpressed with VAPB (1.7 ± 0.1), TM VAPB (1.6 ± 0.2), the MSP domain (MSP VAPB ), the MSP with half of the CC domain (MSP-CC 0.5 VAPB ), or with the complete CC domain (MSP-CC VAPB ). I , J ) Relative current amplitudes of HCN2 HA Ex expressed alone or with TM VAPB (1.3 ± 0.1) ( I ) and the respective changes in the relative surface membrane expression analyzed as RLUs, using a single cell chemiluminescence assay (TM VAPB 1.8 ± 0.2) ( J ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Construct, Expressing, Chemiluminescence Immunoassay

    VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB determines surface expression and dendritic localization of HCN2. A ) Live cell imaging of HeLa cells transfected with an N-terminally EGFP-tagged HCN2 carrying an extracellular HA-epitope ( EGFP HCN2 HA Ex ) alone or cotransfected with VAPB or the TM segment of VAPB (TM VAPB ). B ) Chemiluminescence assays of fixed non-permeabilized HeLa cells, analyzing the surface expression as relative light units (RLUs) for EGFP HCN2 HA Ex alone and after cotransfection with VAPB (1.6 ± 0.1). Upper inset illustrates a representative control Western blot showing an unaltered HCN2 prote in expression. C ) Chemiluminescence surface expression assay as in B , but using TM VAPB (1.6 ± 0.1). D ) Immunocytochemistry of HA VAPB transfected cortical neurons. Endogenous HCN2 (green) is colocalizing (white) with HA VAPB (magenta) in the soma and dendrites. Anti–MAP2-staining illustrating an intact neuronal network and dendrites (blue). E ) Immunocytochemistry experiment as in D , but transfecting the ALS8 mutation HA VAPB P56S (magenta), leading to an aggregation of VAPB P56S in the soma of the neurons. Also, HCN2 fluorescence (green) was focused in the soma and dendritic localization was lost, despite an intact neuronal network (α-MAP2, blue). Scale bars, 20 µm ( A , D , E ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. ** P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Expressing, Live Cell Imaging, Transfection, Cotransfection, Western Blot, Immunocytochemistry, Staining, Mutagenesis, Fluorescence

    Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: Codistribution of VAPs with HCN2 and contribution to thalamic I h . A – E ), Distribution of HCN2, VAPB, and VAPA mRNA in mouse brain and spinal cord. ISH analysis of HCN2, VAPB, and VAPA using DIG-labeled riboprobes, revealing mRNA expression of VAPB in cortical areas ( A ), hippocampus ( B ), thalamus ( C ), cerebellum ( D ) (arrows point to interneurons in the granular layer), and spinal cord ( E ). Note the overlapping distribution of VAPB with HCN2 and VAPA mRNA. Am, amygdala; CA, cornu ammonis; DG, dentate gyrus; DH, dorsal horn; gcl, granule cell layer; Hb, habenulae; ic, internal capsule; LG, lateral geniculate ncl.; m, molecular cell layer; pcl, Purkinje cell layer; RTh, reticular thalamic ncl.; Sth, subthalamic ncl.; VB, ventrobasal thalamus; Th, thalamus; VH, ventral horn. F ) Representative current traces elicited in slice patch-clamp experiments of the ventrobasal thalamus (VB) of wild-type animals (control) and VAPB −/− mice. G ) The I h current was significantly reduced in VAPB −/− mice (15.4 ± 1.1 pA/pF) compared with control animals (22.2 ± 2.3 pA/pF). H ) Average activation curves of the VB I h current for control and VAPB −/− mice. V 1/2 of activation for control (−91.6 ± 1.3 mV, n = 8) and VAPB −/− (−87.5 ± 1.2 mV, n = 7). Scale bars: 500 µm ( A–C , E ), 100 µm ( D ). All data are presented as means ± sem . The number of experiments ( n ) is indicated in the respective bar graphs. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: In Situ Hybridization, Labeling, Expressing, Patch Clamp, Mouse Assay, Activation Assay

    VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Journal: The FASEB Journal

    Article Title: The VAMP-associated protein VAPB is required for cardiac and neuronal pacemaker channel function

    doi: 10.1096/fj.201800246R

    Figure Lengend Snippet: VAPB selectively increases HCN1 and HCN2 currents. A ) Y2H direct interaction assay. Transformation control (-LW), leucine, and tryptophan dropout. Interaction read-out (-LWHA), additional dropout of histidine and adenine. pAL-Alg5, positive control. pPR3-N, negative control. B ) Topology of VAPB. C ) GST VAPB pull-down of HCN2 EGFP using transfected HeLa cells. D ) GST VAPA, GST VAMP1, or GST VAMP2 pull-down of HCN2 EGFP using transfected HeLa cells. E ) GST VAPA pull-down of HCN2 and endogenous VAPB, using HCN2 EGFP transfected HeLa cells. F ) Pull-down of in vitro translated HCN2 (untagged). G ) Pull-down of HCN2 from rat brain lysates. H , I ) Representative currents ( H ) of HCN2 expressed in oocytes alone or with VAPB and the relative current amplitudes ( I ) analyzed over 3 d. J ) Relative currents of HCN1, HCN2, and HCN4 alone or coexpressed with VAPB. K , L ) Relative currents of different potassium channels ( K ) coexpressed with VAPB and of HCN2 ( L ) coexpressed with VAPA, VAPB, or VAPC. M ) Relative currents of HCN2 coexpressed with a mixture of VAPA/B (1:1). N ) Representative macropatch recordings in different configurations: on cell (o.c.), inside-out after patch excision (i.o.), and after application of 100 µM cAMP (i.o.+100 µM cAMP). O , P ) Activation curves for HCN2 alone ( n = 6) ( O ), recorded as in N , or after coexpression with VAPB ( n = 8) ( P ). Q ) V 1/2 values for HCN2 expressed alone or with VAPB in different patch modes. R ) Relative currents of HCN2 HA Ex alone or with VAPB. S ) Relative surface expression of HCN2 HA Ex expressed alone or with VAPB, analyzed as relative light units (RLUs). T ) Relative currents of HCN2 expressed alone or with VAPB or VAPB P56S . All data are presented as means ± sem . The number of cells ( n ) is indicated in the bar graphs. N.s., not significant. * P

    Article Snippet: Untagged HCN2 protein was detected with rabbit α-HCN2 antibody (APC-030, 1:300; Alomone Labs, Jerusalem, Israel) and peroxidase-conjugated goat α-rabbit IgG antibody (32460, 1:2000; Thermo Fisher Scientific) as the secondary antibody.

    Techniques: Transformation Assay, Positive Control, Negative Control, Transfection, In Vitro, Activation Assay, Expressing

    Kcnj10 and Kcnq1 are expressed in the stria vascularis of Atp6v0a4 mutant mice. (A,C) Mid-modiolar sections of the inner ear of control (A) and mutant (C) mice at P20. Regions in boxes in A and C are magnified in B and D, respectively. In wild types and mutants, a similar Kcnj10 expression pattern is observed. Especially strong expression is found in the intermediate cells of the stria vascularis (B,D, black arrowheads), whereas the marginal cells are negative (B,D, red arrowheads). It is also noticeable that mutants examined ( n =5) did not seem to show Reissner's membrane so much distended towards the scala vestibuli as at P0 and P5. We think this might be due to the decalcification process used to facilitate the sectioning of these samples, or to the resolution of any pressure difference across Reissner's membrane by rupture. (E,F) Expression of Kcnq1 is specifically found in the marginal cells of the stria vascularis at P20 in both wild types and mutants (E,F, red arrowheads). RM, Reissner's membrane; SV, stria vascularis. Scale bars: 100 μm (A,C); 20 μm (B,D-F).

    Journal: Disease Models & Mechanisms

    Article Title: Mice deficient in H+-ATPase a4 subunit have severe hearing impairment associated with enlarged endolymphatic compartments within the inner ear

    doi: 10.1242/dmm.010645

    Figure Lengend Snippet: Kcnj10 and Kcnq1 are expressed in the stria vascularis of Atp6v0a4 mutant mice. (A,C) Mid-modiolar sections of the inner ear of control (A) and mutant (C) mice at P20. Regions in boxes in A and C are magnified in B and D, respectively. In wild types and mutants, a similar Kcnj10 expression pattern is observed. Especially strong expression is found in the intermediate cells of the stria vascularis (B,D, black arrowheads), whereas the marginal cells are negative (B,D, red arrowheads). It is also noticeable that mutants examined ( n =5) did not seem to show Reissner's membrane so much distended towards the scala vestibuli as at P0 and P5. We think this might be due to the decalcification process used to facilitate the sectioning of these samples, or to the resolution of any pressure difference across Reissner's membrane by rupture. (E,F) Expression of Kcnq1 is specifically found in the marginal cells of the stria vascularis at P20 in both wild types and mutants (E,F, red arrowheads). RM, Reissner's membrane; SV, stria vascularis. Scale bars: 100 μm (A,C); 20 μm (B,D-F).

    Article Snippet: Antibodies used were: rabbit anti-H+-ATPase subunit a4 (RA2922, 1:400) , and subunit B1 (1:400, a gift from Ge-Hong Sun Wada, Doshisha Women's College, Kyotanabe, Japan), goat anti-Foxi1 (1:50; Abcam), goat anti-Pds (1:50; Santa Cruz Biotechnology), rabbit anti-Kcnj10 (1:300; Alomone) and rabbit anti-Kcnq1 (1:200; Santa Cruz Biotechnology).

    Techniques: Mutagenesis, Mouse Assay, Expressing

    The synthesized GILZ-p inhibited LPS induced Müller cell gliosis. Western blot analysis was performed to determine the protein expression levels of glial fibrillary acidic protein (GFAP) (A,B) , and AQP4 (C,D) in Müller cells treated with 1000 ng/ml LPS in combination with different concentrations of GILZ-p (0.01, 0.1, 1, and 10 μM) for 24 h. β-actin was used as the loading control. The results of quantitative analysis, as determined by densitometric analysis, were expressed as relative to β-actin. Data represent the mean ± SE; the Mann–Whitney U -test was used for comparisons between two groups. n = 3 for each group. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: A Synthesized Glucocorticoid- Induced Leucine Zipper Peptide Inhibits Retinal Müller Cell Gliosis

    doi: 10.3389/fphar.2018.00331

    Figure Lengend Snippet: The synthesized GILZ-p inhibited LPS induced Müller cell gliosis. Western blot analysis was performed to determine the protein expression levels of glial fibrillary acidic protein (GFAP) (A,B) , and AQP4 (C,D) in Müller cells treated with 1000 ng/ml LPS in combination with different concentrations of GILZ-p (0.01, 0.1, 1, and 10 μM) for 24 h. β-actin was used as the loading control. The results of quantitative analysis, as determined by densitometric analysis, were expressed as relative to β-actin. Data represent the mean ± SE; the Mann–Whitney U -test was used for comparisons between two groups. n = 3 for each group. ∗ P

    Article Snippet: The membranes were blocked in 5% non-fat milk at room temperature for 1 h and incubated with the following antibodies: anti-monocyte chemoattractant protein (MCP)-1 (ab25124; Abcam, Cambridge, United Kingdom), anti-intercellular adhesion molecule (ICAM)-1 (ab171123; Proteintech, Chicago, IL, United States), anti-IL1β (ab9787; Abcam), Anti-tumor necrosis factor (TNF)α (PB0270, Boster Biological Technology, Wuhan, China), rabbit anti-p65 polyclonal antibody (ab16502; Abcam), anti-phospho-NF-κB p65 (Ser536) rabbit monoclonal antibody (3033P; Cell Signaling Technology, Beverly, MA, United States), anti-AQP4 (300-314, Alomone Labs, Jerusalem, Israel), anti-glial fibrillary acidic protein (GFAP) (ab10062, Abcam), or rabbit anti β-actin antibody (ab69512; Abcam) overnight.

    Techniques: Synthesized, Western Blot, Expressing, MANN-WHITNEY

    Alteration of GFAP and AQP4 in the cortex of EAE mice. A , Alterations of astrocytic proteins in the cortex at different stages of rrEAE. B , Summary of the alterations of intensities. C , Summary of increased GFAP + cells. (B, C) One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Alteration of GFAP and AQP4 in the cortex of EAE mice. A , Alterations of astrocytic proteins in the cortex at different stages of rrEAE. B , Summary of the alterations of intensities. C , Summary of increased GFAP + cells. (B, C) One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Mouse Assay

    Altered astrocytes in the hippocampus and cortex in Kv3.1 KO mice. The confocal image stacks of hippocampus (A-C) and cortex (D-F) were costained for GFAP (green) and AQP4 (red) from WT (A,D) , AnkG KO (B,E) and Kv3.1 KO (C,F) mice. The collapsed 2D image is on the top and 3 cross sections are at the bottom. The crossbars are centered on astrocyte endfeet with colocalizing AQP4 and GFAP. Scale bars, 100 μm.

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Altered astrocytes in the hippocampus and cortex in Kv3.1 KO mice. The confocal image stacks of hippocampus (A-C) and cortex (D-F) were costained for GFAP (green) and AQP4 (red) from WT (A,D) , AnkG KO (B,E) and Kv3.1 KO (C,F) mice. The collapsed 2D image is on the top and 3 cross sections are at the bottom. The crossbars are centered on astrocyte endfeet with colocalizing AQP4 and GFAP. Scale bars, 100 μm.

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Mouse Assay

    Differentially altered expression of astrocytic proteins in the cerebellum of EAE mice. A , The confocal image stack of cerebellar molecular layer that was stained for GFAP (green), AQP4 (red) and Vim (blue) from a control mouse. Collapsed 2D image is on the left and 3 cross sections of 3D are on the right. B , The images at the peak stage of an rrEAE mouse. C , The images at the remitting stage of an rrEAE mouse. D , The images at the relapsing stage of an rrEAE mouse. E , The confocal images of cerebellar WM from a Thy1-YFP transgenic mouse. YFP (green), AQP4 (red) and GFAP (blue). In (A-E) , the crossbars are centered on astrocytic endfeet with colocalizing AQP4 and GFAP. F , The confocal images at the peak stage of chEAE. The crossbars show the lesion edge with upregulated AQP4 and GFAP. G , Structural diagram of cerebellar cortex. H , Summary of changes of protein levels during rrEAE in cerebellar molecular layer. One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Differentially altered expression of astrocytic proteins in the cerebellum of EAE mice. A , The confocal image stack of cerebellar molecular layer that was stained for GFAP (green), AQP4 (red) and Vim (blue) from a control mouse. Collapsed 2D image is on the left and 3 cross sections of 3D are on the right. B , The images at the peak stage of an rrEAE mouse. C , The images at the remitting stage of an rrEAE mouse. D , The images at the relapsing stage of an rrEAE mouse. E , The confocal images of cerebellar WM from a Thy1-YFP transgenic mouse. YFP (green), AQP4 (red) and GFAP (blue). In (A-E) , the crossbars are centered on astrocytic endfeet with colocalizing AQP4 and GFAP. F , The confocal images at the peak stage of chEAE. The crossbars show the lesion edge with upregulated AQP4 and GFAP. G , Structural diagram of cerebellar cortex. H , Summary of changes of protein levels during rrEAE in cerebellar molecular layer. One-Way ANOVA followed by Fisher’s test. *, significant difference from Control for each antibody, p

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Expressing, Mouse Assay, Staining, Transgenic Assay

    Activation of astrocytes in the hippocampus of EAE mice. A , Increased GFAP but not AQP4 staining in the hippocampus of EAE mice. B , Increased Vim staining in the hippocampus during EAE progression. C , Enlarged images of individual astrocytes in the hippocampus clearly show the increase of Vim staining. High magnification confocal image stacks were obtained from control (D) and EAE (E) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. The crossbars reveal astrocytic endfeet with colocalizing AQP4 and GFAP. F , Summary of the levels of astrocytic proteins at different stages during EAE progression. One-Way ANOVA followed by Fisher’s test, *, significant difference from Control for each antibody, p

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Activation of astrocytes in the hippocampus of EAE mice. A , Increased GFAP but not AQP4 staining in the hippocampus of EAE mice. B , Increased Vim staining in the hippocampus during EAE progression. C , Enlarged images of individual astrocytes in the hippocampus clearly show the increase of Vim staining. High magnification confocal image stacks were obtained from control (D) and EAE (E) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. The crossbars reveal astrocytic endfeet with colocalizing AQP4 and GFAP. F , Summary of the levels of astrocytic proteins at different stages during EAE progression. One-Way ANOVA followed by Fisher’s test, *, significant difference from Control for each antibody, p

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Activation Assay, Mouse Assay, Staining, Transgenic Assay

    Upregulation of GFAP and AQP4 in the cerebellum in AnkG and Kv3.1 KO mice. A , High magnification image stacks of cerebellar molecular layer stained with anti-PKCγ (green) and anti-GFAP (red) antibodies. The collapsed 2D image is on the left, and 3 cross sections are on the right. B , Confocal image stacks from the AnkG KO mice. C , Confocal image stacks from the Kv3.1 KO mice. In (A,C) , the crossbars reveal radially oriented GFAP + Bergmann glial processes in (A) WT and (C) Kv3.1 KO mice. In (B) , the crossbars are centered on highly upregulated GFAP + astrocyte processes in the absence of Purkinje neurons in an AnkG KO mouse. D , A single confocal image of the granule cell layer in a WT mouse. E , An image from the AnkG KO mice. F , An image from the Kv3.1 KO mice. G , Normalized fluorescence intensity in the molecular layer. H , Normalized fluorescence intensity in the granule cell layer. (G, H) One-Way ANOVA followed by Fisher’s test. *, significant difference from Wildtype for each antibody, p

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Upregulation of GFAP and AQP4 in the cerebellum in AnkG and Kv3.1 KO mice. A , High magnification image stacks of cerebellar molecular layer stained with anti-PKCγ (green) and anti-GFAP (red) antibodies. The collapsed 2D image is on the left, and 3 cross sections are on the right. B , Confocal image stacks from the AnkG KO mice. C , Confocal image stacks from the Kv3.1 KO mice. In (A,C) , the crossbars reveal radially oriented GFAP + Bergmann glial processes in (A) WT and (C) Kv3.1 KO mice. In (B) , the crossbars are centered on highly upregulated GFAP + astrocyte processes in the absence of Purkinje neurons in an AnkG KO mouse. D , A single confocal image of the granule cell layer in a WT mouse. E , An image from the AnkG KO mice. F , An image from the Kv3.1 KO mice. G , Normalized fluorescence intensity in the molecular layer. H , Normalized fluorescence intensity in the granule cell layer. (G, H) One-Way ANOVA followed by Fisher’s test. *, significant difference from Wildtype for each antibody, p

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Mouse Assay, Staining, Fluorescence

    Activation of astrocytes in spinal cord white matter. A , Clinical scores (top) and body weight (bottom) of mice with chEAE. B , White matter (WM) and gray matter (GM) in spinal cord longitudinal section was stained with FMG (green) and nuclear dye (blue). Box 1 shows both GM and WM and box 2 shows only WM. Spinal cord sections, control (C) and EAE peak (D) , were co-stained for GFAP (green, top), AQP4 (red), Hoechst (blue), and FMG (green, bottom). Co-staining of Kv1.4 (green, top), Vim (red), Hoechst (blue) and FMG (green, bottom) were also performed on control (E) and EAE (F) spinal cord sections. High magnification confocal image stacks were obtained from control (G) and EAE (H) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. In (G) , the crossbars are centered on a putative node of Ranvier. In (H) , the crossbars are centered on the AQP4+/GFAP + lesion edge. Scale bars, 500 μm in C-F , 50 μm in G , H .

    Journal: Acta Neuropathologica Communications

    Article Title: Astrocytes differentially respond to inflammatory autoimmune insults and imbalances of neural activity

    doi: 10.1186/2051-5960-1-70

    Figure Lengend Snippet: Activation of astrocytes in spinal cord white matter. A , Clinical scores (top) and body weight (bottom) of mice with chEAE. B , White matter (WM) and gray matter (GM) in spinal cord longitudinal section was stained with FMG (green) and nuclear dye (blue). Box 1 shows both GM and WM and box 2 shows only WM. Spinal cord sections, control (C) and EAE peak (D) , were co-stained for GFAP (green, top), AQP4 (red), Hoechst (blue), and FMG (green, bottom). Co-staining of Kv1.4 (green, top), Vim (red), Hoechst (blue) and FMG (green, bottom) were also performed on control (E) and EAE (F) spinal cord sections. High magnification confocal image stacks were obtained from control (G) and EAE (H) Thy1-YFP transgenic mice. Images contain YFP (green), GFAP (blue) and AQP4 (red). The collapsed 2D image is on the left, and 3 cross sections are on the right. In (G) , the crossbars are centered on a putative node of Ranvier. In (H) , the crossbars are centered on the AQP4+/GFAP + lesion edge. Scale bars, 500 μm in C-F , 50 μm in G , H .

    Article Snippet: The following antibodies were used in our study: rabbit polyclonal anti-Kv1.4, anti-AQP4 (Alomone Labs, Jerusalem, Israel), and anti-PKCγ (Santa Cruz Biotechnology, Dallas, TX); goat polyclonal anti-GFAP (AbCAM, Cambridge, MA, USA); chicken polyclonal anti-Vimentin (Millipore, Temecula, CA); and Dylight 488-, Dylight 649-, Cy3-, and Cy5-conjugated secondary antibodies (Jackson Immuno Research Laboratories, West Grove, PA, USA).

    Techniques: Activation Assay, Mouse Assay, Staining, Transgenic Assay