glua2  (Alomone Labs)


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  • 80
    Name:
    GluR2 GluA2 extracellular Blocking Peptide
    Description:
    A Blocking Peptide for Anti GluR2 GluA2 extracellular Antibody
    Catalog Number:
    BLP-GC005
    Price:
    245.0
    Category:
    Control reagent Negative Control
    Source:
    Synthetic peptide
    Applications:
    Blocking
    Purity:
    >70% (amino acid analysis and mass spectrometry).
    Size:
    40 mcg
    Format:
    Lyophilized powder
    Buy from Supplier


    Structured Review

    Alomone Labs glua2
    GluR2 GluA2 extracellular Blocking Peptide
    A Blocking Peptide for Anti GluR2 GluA2 extracellular Antibody
    https://www.bioz.com/result/glua2/product/Alomone Labs
    Average 80 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glua2 - by Bioz Stars, 2021-10
    80/100 stars

    Images

    1) Product Images from "α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly"

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    Journal: Cell reports

    doi: 10.1016/j.celrep.2021.109396

    α2δ-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p
    Figure Legend Snippet: α2δ-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p

    Techniques Used: Expressing, Plasmid Preparation

    α2δ-1 disrupts the heteromeric assembly of GluA1/GluA2 and increases GluA2 retention in the endoplasmic reticulum (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p
    Figure Legend Snippet: α2δ-1 disrupts the heteromeric assembly of GluA1/GluA2 and increases GluA2 retention in the endoplasmic reticulum (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p

    Techniques Used:

    α2δ-1 physically interacts with GluA1 and GluA2 in vitro and in vivo (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p
    Figure Legend Snippet: α2δ-1 physically interacts with GluA1 and GluA2 in vitro and in vivo (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p

    Techniques Used: In Vitro, In Vivo, Co-Immunoprecipitation Assay, Plasmid Preparation, Ligation, Construct

    Gabapentin and the α2δ-1CT-Tat peptide normalize synaptic expression of GluA2-containing AMPARs in the spinal cord diminished in neuropathic pain (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p
    Figure Legend Snippet: Gabapentin and the α2δ-1CT-Tat peptide normalize synaptic expression of GluA2-containing AMPARs in the spinal cord diminished in neuropathic pain (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p

    Techniques Used: Expressing

    Gabapentin and the α2δ-1CT-Tat C terminus peptide restore heteromeric GluA1/GluA2 receptors diminished by α2δ-1 coexpression (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p
    Figure Legend Snippet: Gabapentin and the α2δ-1CT-Tat C terminus peptide restore heteromeric GluA1/GluA2 receptors diminished by α2δ-1 coexpression (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p

    Techniques Used: Transfection

    Related Articles

    FLAG-tag:

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly
    Article Snippet: .. Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich). ..

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    Alomone Labs anti glur2 glua2 extracellular antibody
    α2δ-1 reduces surface and synaptic expression of <t>GluA2</t> and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p
    Anti Glur2 Glua2 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti glur2 glua2 extracellular antibody/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    94
    Alomone Labs anti glua2
    Mapping the TARP γ-2 Contact Region on <t>GluA2</t> (A) coIP of GluA2 variants with TARP γ-2. The blot was probed with polyclonal GluA2 antibody (top panel) and anti γ-2 (bottom panel). Both WT and ΔNTD protein migrated as monomer (M) and dimer (D), denoted by arrowheads. Note that while inputs were comparable, amounts of IPed GluA2 varied between conditions. (B) Schematic of the peptide array layout (right). Each peptide is spotted onto a nitrocellulose membrane (C). Peptide coverage of the rat GluA2 sequence is outlined in the left panel in color code as indicated. The four GluA2 regions—the NTD lower lobe, the NTD-LBD linkers, the LBD, and the TM segments of the channel—are highlighted. GluA2 peptide numbers covering each domain are indicated in brackets. See Table S1 for peptide sequences. (C) Regions of GluA2 binding to TARP γ-2. (Upper panel) Nonspecific signal, resulting from anti-γ-2 antibody binding to GluA2 in the absence of the γ-2 probe (“AB control”). AMPAR domains are highlighted in boxes and match the color scheme in (B). Peptide numbers are indicated on the side. The membrane was exposed to an X-ray film for 2 min; the arrow denotes nonspecific signals. (Lower panel) The same membrane was probed with full-length TARP γ-2 and detected with anti-γ-2 AB followed by a HRP-labeled secondary AB (2 min exposure). Individual AMPAR secondary structure elements, corresponding to NTD and LBD helices, are highlighted in stippled boxes on the blot (compared with D). The bottom panel shows a longer exposure for the LBD-A1 region (yellow) and the NTD-LBD linker (blue). See Figure S4 A for a longer exposure of the blot. (D) TARP binding sites deduced from the peptide array in (C) are mapped onto the extracellular region of GluA2 (PDB: 3KG2 ). NTD interaction sites are denoted in deep red (strong interaction) and light pink (weaker interaction; see graded bar below), with alpha helices contacted by γ-2 denoted by (D), (F), and (H). LBD interaction sites are highlighted in brown (strong interaction) and yellow (weaker interaction). The three core contact regions, A–C, are denoted. Region A spans the glutamate binding cleft (interaction sites A 1 and A 2 ); region B encompasses the LBD-TM linker 1, and region C corresponds to the flip-flop cassette (denoted with a stippled ellipsoid). See also Figures S3 , S4 , and S6 and Table S1 .
    Anti Glua2, 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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    Alomone Labs rabbit antibody anti glua2
    Vezatin concentrates in spines during neuronal differentiation. A , Developmental distribution of Vezatin showing its concentration in growth cone and dendrites early during neuronal differentiation (inset, left) and in dendrites and spines in maturing and mature neurons (insets, middle and right). B , At the early stage of neuronal differentiation (left) and later, at the onset of dendritic spine development, Vezatin does not colocalize with presynaptic Syb2 (right, arrow). C , Postsynaptic Vezatin is partially codistributed with transsynaptic N-cadherin. C1 , The arrowhead and the arrow are pointing to a spine where Vezatin is (yellow) and is not (red aside green) clearly overlapping the N-cadherin signal, respectively. D , Vezatin is expressed in the soma of interneurons stained with GA D6 7 (left). Vezatin does not codistribute with GA D6 5 at the axonal compartment (right). MAP2 is used to label dendrite. E , Snapshot of a time-lapse image of a living cortical neuron transfected with Vezatin-GFP and RFP-actin showing codistribution in a hemicircumferential ring in a growth cone. Top, inset, DIV 3, fixed cultured hippocampal neuron immunostained for Vezatin and <t>GluA2,</t> a glutamate receptor, showing that Vezatin expression is restricted to an arc-shaped region of the growth cone. F , Vezatin colocalizes with phalloidin (F-actin) in dendrites and in developing spines at early synaptic sites (DIV 14). Scale bars, 10 μm. G , Protein expression in mouse hippocampi extracts. The anti-Vezatin antibody recognized the two major Vezatin isoforms (see text) with band intensities approximately 2× weaker in the heterozygous floxed/null ( fl/null ) compared to the floxed/floxed ( fl/fl ) extract. Other few alternatively spliced variants are expressed in minor quantities. Loading control, GAPDH.
    Rabbit Antibody Anti Glua2, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs nbqx
    Gliosis within the hippocampus and dentate gyrus of antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, <t>GYKI-52466</t> and <t>NBQX</t> or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were sacrificed on day 21 post infection. Gliosis was scored as described in the Methods. The number of mice in each group is given as N above each column. Data is given as mean + SEM. †p
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    α2δ-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p

    Journal: Cell reports

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    doi: 10.1016/j.celrep.2021.109396

    Figure Lengend Snippet: α2δ-1 reduces surface and synaptic expression of GluA2 and heteromeric GluA1/GluA2 receptors (A) Original blots and quantification show the protein levels of GluA1 and GluA2 in HEK293 cells cotransfected with GluA1/GluA2 and control vector (pcDNA3, P3), α2δ-1, α2δ-2, or α2δ-3 (n = 4 per group). ***p

    Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

    Techniques: Expressing, Plasmid Preparation

    α2δ-1 disrupts the heteromeric assembly of GluA1/GluA2 and increases GluA2 retention in the endoplasmic reticulum (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p

    Journal: Cell reports

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    doi: 10.1016/j.celrep.2021.109396

    Figure Lengend Snippet: α2δ-1 disrupts the heteromeric assembly of GluA1/GluA2 and increases GluA2 retention in the endoplasmic reticulum (A and B) Original blots (A) and quantification (B) show that α2δ-1 coexpression diminishes heteromeric GluA1/GluA2 receptors in the ER of HEK293 cells. n = 5 per group. *p

    Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

    Techniques:

    α2δ-1 physically interacts with GluA1 and GluA2 in vitro and in vivo (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p

    Journal: Cell reports

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    doi: 10.1016/j.celrep.2021.109396

    Figure Lengend Snippet: α2δ-1 physically interacts with GluA1 and GluA2 in vitro and in vivo (A) CoIP shows the interaction between α2δ-1 and GluA1 and GluA2 in HEK293 cells. Cells cotransfected with GFP-tagged α2δ-1 and GluA1, GluA2, GluA1/GluA2, or FLAG-stargazin (STG). (B) CoIP shows the interaction of α2δ-1 with homomeric GluA1 or GluA2 in HEK293 cells. P3, control vector. (C) CoIP shows the interaction of α2δ-1 with heteromeric GluA1/GluA2 in HEK293 cells. (D) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 in the dorsal spinal cord of rats subjected to a sham procedure (S) or spinal nerve ligation (L). (E) CoIP shows the interaction of α2δ-1 with GluA1 and GluA2 subunits in the normal spinal cord tissue of two human donors (S1 and S2). (F) α2δ-1 interacts with GluA1 and GluA2 subunits via its C terminus. HEK293 cells were cotransfected with GluA1/GluA2 and various PC-tagged α2δ-1 constructs. δ-1ΔCT, δ-1 without the C terminus; CT, the C terminus of δ-1; VWA, von Willebrand factor type A domain. (G) CoIP shows that α2δ-1CT-Tat peptide disrupts the α2δ-1 interaction with GluA1 and GluA2 in HEK293 cells. Cell were cotransfected with GluA1/GluA2 and α2δ-1 or FLAG-α2δ-1 and were treated with 1 μM α2δ-1CT-Tat peptide (Pept) or 1 μM Tat-fused Cont peptide for 30 min. ***p

    Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

    Techniques: In Vitro, In Vivo, Co-Immunoprecipitation Assay, Plasmid Preparation, Ligation, Construct

    Gabapentin and the α2δ-1CT-Tat peptide normalize synaptic expression of GluA2-containing AMPARs in the spinal cord diminished in neuropathic pain (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p

    Journal: Cell reports

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    doi: 10.1016/j.celrep.2021.109396

    Figure Lengend Snippet: Gabapentin and the α2δ-1CT-Tat peptide normalize synaptic expression of GluA2-containing AMPARs in the spinal cord diminished in neuropathic pain (A and B) Original blots and quantification show the effect of gabapentin and α2δ-1CT-Tat peptide on the protein levels of GluA1 and GluA2 in the spinal cord synaptosome (A) and the ER (B) of sham and SNL rats (n = 6 rats per group). Spinal cord slices were treated with vehicle (Cont), 100 μM GBP, 1 μM control peptide (P(−)), or 1 μM α2δ-1CT-Tat peptide (P(+)). **p

    Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

    Techniques: Expressing

    Gabapentin and the α2δ-1CT-Tat C terminus peptide restore heteromeric GluA1/GluA2 receptors diminished by α2δ-1 coexpression (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p

    Journal: Cell reports

    Article Title: α2δ-1 switches the phenotype of synaptic AMPA receptors by physically disrupting heteromeric subunit assembly

    doi: 10.1016/j.celrep.2021.109396

    Figure Lengend Snippet: Gabapentin and the α2δ-1CT-Tat C terminus peptide restore heteromeric GluA1/GluA2 receptors diminished by α2δ-1 coexpression (A and B) Original GCaMP images and signals show intracellular Ca 2+ changes in response to 5 mM glutamate (Glut) in HEK293 cells transfected with GluA1/GluA2 (A) or GluA1/GluA2/α2δ-1 (B). (C) Mean data show effects of treatment with vehicle (n = 54 cells), gabapentin (100 μM, n = 28 cells), α2δ-1CT-Tat peptide (1 μM, n = 26 cells), or control peptide (1 μM, n = 21 cells) on the ratio (ΔF/F0) of GCaMP signals elicited by glutamate. ***p

    Article Snippet: Samples were immunoblotted after being washed three times with IP buffer. α2δ-1 was detected using rabbit anti-α2δ-1 antibody (#C5105, 1:1,000; Sigma-Aldrich), GluA1 was detected using mouse anti-GluA1 antibody (#75-327, 1:1,000; NeuroMab) or rabbit anti-GluA1 antibody (#ACC-015, 1:1,000; Alomone Labs), GluA2 was detected by using mouse anti-GluA2 antibody (#75-002, 1:1,000; NeuroMab) or rabbit anti-GluA2 antibody (#AGC-005, 1:1,000; Alomone Labs), and Flag Tag was detected using mouse anti-Flag antibody (#F1804, 1:1,000; Sigma-Aldrich) or rabbit anti-Flag antibody (#F7425, 1:1,000; Sigma-Aldrich).

    Techniques: Transfection

    Mapping the TARP γ-2 Contact Region on GluA2 (A) coIP of GluA2 variants with TARP γ-2. The blot was probed with polyclonal GluA2 antibody (top panel) and anti γ-2 (bottom panel). Both WT and ΔNTD protein migrated as monomer (M) and dimer (D), denoted by arrowheads. Note that while inputs were comparable, amounts of IPed GluA2 varied between conditions. (B) Schematic of the peptide array layout (right). Each peptide is spotted onto a nitrocellulose membrane (C). Peptide coverage of the rat GluA2 sequence is outlined in the left panel in color code as indicated. The four GluA2 regions—the NTD lower lobe, the NTD-LBD linkers, the LBD, and the TM segments of the channel—are highlighted. GluA2 peptide numbers covering each domain are indicated in brackets. See Table S1 for peptide sequences. (C) Regions of GluA2 binding to TARP γ-2. (Upper panel) Nonspecific signal, resulting from anti-γ-2 antibody binding to GluA2 in the absence of the γ-2 probe (“AB control”). AMPAR domains are highlighted in boxes and match the color scheme in (B). Peptide numbers are indicated on the side. The membrane was exposed to an X-ray film for 2 min; the arrow denotes nonspecific signals. (Lower panel) The same membrane was probed with full-length TARP γ-2 and detected with anti-γ-2 AB followed by a HRP-labeled secondary AB (2 min exposure). Individual AMPAR secondary structure elements, corresponding to NTD and LBD helices, are highlighted in stippled boxes on the blot (compared with D). The bottom panel shows a longer exposure for the LBD-A1 region (yellow) and the NTD-LBD linker (blue). See Figure S4 A for a longer exposure of the blot. (D) TARP binding sites deduced from the peptide array in (C) are mapped onto the extracellular region of GluA2 (PDB: 3KG2 ). NTD interaction sites are denoted in deep red (strong interaction) and light pink (weaker interaction; see graded bar below), with alpha helices contacted by γ-2 denoted by (D), (F), and (H). LBD interaction sites are highlighted in brown (strong interaction) and yellow (weaker interaction). The three core contact regions, A–C, are denoted. Region A spans the glutamate binding cleft (interaction sites A 1 and A 2 ); region B encompasses the LBD-TM linker 1, and region C corresponds to the flip-flop cassette (denoted with a stippled ellipsoid). See also Figures S3 , S4 , and S6 and Table S1 .

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: Mapping the TARP γ-2 Contact Region on GluA2 (A) coIP of GluA2 variants with TARP γ-2. The blot was probed with polyclonal GluA2 antibody (top panel) and anti γ-2 (bottom panel). Both WT and ΔNTD protein migrated as monomer (M) and dimer (D), denoted by arrowheads. Note that while inputs were comparable, amounts of IPed GluA2 varied between conditions. (B) Schematic of the peptide array layout (right). Each peptide is spotted onto a nitrocellulose membrane (C). Peptide coverage of the rat GluA2 sequence is outlined in the left panel in color code as indicated. The four GluA2 regions—the NTD lower lobe, the NTD-LBD linkers, the LBD, and the TM segments of the channel—are highlighted. GluA2 peptide numbers covering each domain are indicated in brackets. See Table S1 for peptide sequences. (C) Regions of GluA2 binding to TARP γ-2. (Upper panel) Nonspecific signal, resulting from anti-γ-2 antibody binding to GluA2 in the absence of the γ-2 probe (“AB control”). AMPAR domains are highlighted in boxes and match the color scheme in (B). Peptide numbers are indicated on the side. The membrane was exposed to an X-ray film for 2 min; the arrow denotes nonspecific signals. (Lower panel) The same membrane was probed with full-length TARP γ-2 and detected with anti-γ-2 AB followed by a HRP-labeled secondary AB (2 min exposure). Individual AMPAR secondary structure elements, corresponding to NTD and LBD helices, are highlighted in stippled boxes on the blot (compared with D). The bottom panel shows a longer exposure for the LBD-A1 region (yellow) and the NTD-LBD linker (blue). See Figure S4 A for a longer exposure of the blot. (D) TARP binding sites deduced from the peptide array in (C) are mapped onto the extracellular region of GluA2 (PDB: 3KG2 ). NTD interaction sites are denoted in deep red (strong interaction) and light pink (weaker interaction; see graded bar below), with alpha helices contacted by γ-2 denoted by (D), (F), and (H). LBD interaction sites are highlighted in brown (strong interaction) and yellow (weaker interaction). The three core contact regions, A–C, are denoted. Region A spans the glutamate binding cleft (interaction sites A 1 and A 2 ); region B encompasses the LBD-TM linker 1, and region C corresponds to the flip-flop cassette (denoted with a stippled ellipsoid). See also Figures S3 , S4 , and S6 and Table S1 .

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Co-Immunoprecipitation Assay, Peptide Microarray, Sequencing, Binding Assay, Labeling

    Mapping TARP γ-2 and TARP γ-8 Residues Involved in AMPAR Interaction (A) Alignment of rat type 1a (γ-2, γ-3) and type 1b (γ-4, γ-8) TARP extracellular loops, Ex1 and Ex2. Conserved residues are shaded brown, and residues highly conserved throughout the Cacng family (γ-1 to γ-8) are boxed in gray. The four cysteines are highlighted in yellow. Curly brackets above the γ-2 (green) and below γ-8 alignment (blue) indicate regions in the center of Ex1 interacting with the AMPAR extracellular domains (NTD and LBD). (B) TARP array encompassing the Ex1 and Ex2 segments of γ-2 (green box) and γ-8 (blue box) probed with the NTD. (Upper panel) Nonspecific signal, resulting from direct anti-GluA2 antibody binding to the membrane (AB control). The Ex1 and Ex2 regions for both TARPs are denoted (dashed line). (Lower panel) the same membrane was exposed to the rat GluA2 NTD followed by probing with anti-GluA2 AB. The membrane exposure time is as indicated. See Table S2 for peptide sequences. (C) γ-2 and γ-8 arrays probed with GluA2 LBD and GluK2 LBD. The negative controls with anti-FLAG AB did not show any signal and thus are not shown. Membranes were then incubated with FLAG-tagged GluA2 LBD or GluK2 LBD and probed with anti-FLAG AB. (Upper panel) GluA2 LBD interaction with γ-2 (same peptides as in Figure 5 B, green box). (Central panel) GluA2 LBD interaction with γ-8 (same peptides as in B, blue box). (Lower panel) the γ-2 membrane previously probed with GluA2 LBD (top panel) was regenerated and probed with the FLAG-tagged GluK2 LBD, which produced no clear binding. Regeneration of this membrane resulted in a clear binding pattern when reprobed with the GluA2 LBD that matched the one shown in (C, top). (D) Schematic representation of TARP structure with the GluA2 NTD and LBD interacting parts of the Ex1 and Ex2 loops highlighted in orange and the highly conserved GLWR motif indicated.

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: Mapping TARP γ-2 and TARP γ-8 Residues Involved in AMPAR Interaction (A) Alignment of rat type 1a (γ-2, γ-3) and type 1b (γ-4, γ-8) TARP extracellular loops, Ex1 and Ex2. Conserved residues are shaded brown, and residues highly conserved throughout the Cacng family (γ-1 to γ-8) are boxed in gray. The four cysteines are highlighted in yellow. Curly brackets above the γ-2 (green) and below γ-8 alignment (blue) indicate regions in the center of Ex1 interacting with the AMPAR extracellular domains (NTD and LBD). (B) TARP array encompassing the Ex1 and Ex2 segments of γ-2 (green box) and γ-8 (blue box) probed with the NTD. (Upper panel) Nonspecific signal, resulting from direct anti-GluA2 antibody binding to the membrane (AB control). The Ex1 and Ex2 regions for both TARPs are denoted (dashed line). (Lower panel) the same membrane was exposed to the rat GluA2 NTD followed by probing with anti-GluA2 AB. The membrane exposure time is as indicated. See Table S2 for peptide sequences. (C) γ-2 and γ-8 arrays probed with GluA2 LBD and GluK2 LBD. The negative controls with anti-FLAG AB did not show any signal and thus are not shown. Membranes were then incubated with FLAG-tagged GluA2 LBD or GluK2 LBD and probed with anti-FLAG AB. (Upper panel) GluA2 LBD interaction with γ-2 (same peptides as in Figure 5 B, green box). (Central panel) GluA2 LBD interaction with γ-8 (same peptides as in B, blue box). (Lower panel) the γ-2 membrane previously probed with GluA2 LBD (top panel) was regenerated and probed with the FLAG-tagged GluK2 LBD, which produced no clear binding. Regeneration of this membrane resulted in a clear binding pattern when reprobed with the GluA2 LBD that matched the one shown in (C, top). (D) Schematic representation of TARP structure with the GluA2 NTD and LBD interacting parts of the Ex1 and Ex2 loops highlighted in orange and the highly conserved GLWR motif indicated.

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Binding Assay, Incubation, Produced

    The Δ Link Mutation Accelerates Recovery from Desensitization in the Presence of TARPs (A) Representative traces illustrating recovery from desensitization (averages of three trials in each case). A 100 ms pulse of 10 mM L-glutamate was followed, at increasing intervals, by a 10 ms test pulse, and the recovery in the amplitude of the test response was fitted by a monoexponential function (dashed lines). Currents were normalized to the first peak, and for clarity, only selected traces are shown. (B) Summary of the data presented in (A). Relative currents at individual time points are shown ± SEM (error bars masked by the symbols). The solid lines are monoexponential fits of the average values (giving time constants of 7.3, 21.8, and 75.6 ms for GluA2 Δ link + γ-2, GluA2 WT + γ-2, and GluA2 WT + γ-8, respectively). (C) Pooled data for the time constant of recovery from desensitization (τ rec ) for GluA2iQ WT and Δ link expressed alone (n = 11 and 8, respectively) or with TARPs γ-2 (n = 17 and 9), γ-3 (n = 6 and 5), γ-4 (n = 8 each), or γ-8 (n = 7 and 8) (shown ± SEM). Two-way ANOVA showed significant main effects of TARP subtype ( F 4, 77 = 62.61, p = 1.91 × 10 −23 ) and linker mutation ( F 1, 77 = 25.58, p = 4.14 × 10 −6 ) and a significant interaction between linker and TARP effects ( F 4, 77 = 3.14, p = 0.019). Asterisks denote significance of difference between WT and Δ link for each TARP condition ( ∗ p

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: The Δ Link Mutation Accelerates Recovery from Desensitization in the Presence of TARPs (A) Representative traces illustrating recovery from desensitization (averages of three trials in each case). A 100 ms pulse of 10 mM L-glutamate was followed, at increasing intervals, by a 10 ms test pulse, and the recovery in the amplitude of the test response was fitted by a monoexponential function (dashed lines). Currents were normalized to the first peak, and for clarity, only selected traces are shown. (B) Summary of the data presented in (A). Relative currents at individual time points are shown ± SEM (error bars masked by the symbols). The solid lines are monoexponential fits of the average values (giving time constants of 7.3, 21.8, and 75.6 ms for GluA2 Δ link + γ-2, GluA2 WT + γ-2, and GluA2 WT + γ-8, respectively). (C) Pooled data for the time constant of recovery from desensitization (τ rec ) for GluA2iQ WT and Δ link expressed alone (n = 11 and 8, respectively) or with TARPs γ-2 (n = 17 and 9), γ-3 (n = 6 and 5), γ-4 (n = 8 each), or γ-8 (n = 7 and 8) (shown ± SEM). Two-way ANOVA showed significant main effects of TARP subtype ( F 4, 77 = 62.61, p = 1.91 × 10 −23 ) and linker mutation ( F 1, 77 = 25.58, p = 4.14 × 10 −6 ) and a significant interaction between linker and TARP effects ( F 4, 77 = 3.14, p = 0.019). Asterisks denote significance of difference between WT and Δ link for each TARP condition ( ∗ p

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Mutagenesis

    Gating Changes Are Mediated by Specific Structural Features of the NTD Linker (A) Sequence of the GluA2 NTD-LBD linker with the glycosylation sites and the amino acid quadruplets deleted in these experiments highlighted in red and blue. (B) Pooled data (mean ± SEM) showing the effects on desensitization (τ w,des ) of NTD-LBD linker mutations in GluA2i in presence of γ-2. LPSG denotes a construct with these four amino acids deleted; LPSG-D combines this with the N385D mutation, and LPSG-D-Q additionally includes N392Q. Following one-way ANOVA ( F 7, 25.7 = 14.69, p = 1.52 × 10 −7 ), pairwise comparisons showed that τ w,des was slower for LPSG (n = 11), LPSG-D (n = 6), LPSG-D-Q (n = 16), and Δ link (n = 7) compared with WT (n = 11) ( ∗∗ p

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: Gating Changes Are Mediated by Specific Structural Features of the NTD Linker (A) Sequence of the GluA2 NTD-LBD linker with the glycosylation sites and the amino acid quadruplets deleted in these experiments highlighted in red and blue. (B) Pooled data (mean ± SEM) showing the effects on desensitization (τ w,des ) of NTD-LBD linker mutations in GluA2i in presence of γ-2. LPSG denotes a construct with these four amino acids deleted; LPSG-D combines this with the N385D mutation, and LPSG-D-Q additionally includes N392Q. Following one-way ANOVA ( F 7, 25.7 = 14.69, p = 1.52 × 10 −7 ), pairwise comparisons showed that τ w,des was slower for LPSG (n = 11), LPSG-D (n = 6), LPSG-D-Q (n = 16), and Δ link (n = 7) compared with WT (n = 11) ( ∗∗ p

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Sequencing, Construct, Mutagenesis

    The NTD-LBD Linker Influences TARP-Dependent Changes in AMPAR Gating (A) Structure of the extracellular region of a GluA2 subunit, showing the NTD (gray), linker (blue), and LBD (yellow) (adapted from PDB: 3KG2). Sequence alignment of the rat GluA1-4 NTD linkers, red residues have been mutated in Δ link with VTxxxLPSG deleted and the two asparagines (N385 and N392 bold, underlined) mutated to Asp and Gln, respectively (analogous to PDB 3KG2). Model of a complete, stretched NTD linker is shown beneath the alignment. (B) Representative normalized current responses evoked by 100 ms glutamate application (gray bars). Outside-out patches were pulled from cells transfected with GluA2iQ WT or Δ link in the absence or presence of γ-2 and the decay of the current (−60 mV) analyzed to determine the time constant of desensitization and the magnitude of the steady state component. (Inset) Pooled data (mean ± SEM) showing the difference in charge transfer (normalized to the peak) during the 100 ms glutamate application ( ∗∗∗ p

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: The NTD-LBD Linker Influences TARP-Dependent Changes in AMPAR Gating (A) Structure of the extracellular region of a GluA2 subunit, showing the NTD (gray), linker (blue), and LBD (yellow) (adapted from PDB: 3KG2). Sequence alignment of the rat GluA1-4 NTD linkers, red residues have been mutated in Δ link with VTxxxLPSG deleted and the two asparagines (N385 and N392 bold, underlined) mutated to Asp and Gln, respectively (analogous to PDB 3KG2). Model of a complete, stretched NTD linker is shown beneath the alignment. (B) Representative normalized current responses evoked by 100 ms glutamate application (gray bars). Outside-out patches were pulled from cells transfected with GluA2iQ WT or Δ link in the absence or presence of γ-2 and the decay of the current (−60 mV) analyzed to determine the time constant of desensitization and the magnitude of the steady state component. (Inset) Pooled data (mean ± SEM) showing the difference in charge transfer (normalized to the peak) during the 100 ms glutamate application ( ∗∗∗ p

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Sequencing, Transfection

    Effects of Ex1 Mutations in TARP γ-2 (A) Sequence of the γ-2 Ex1 region surrounding the highly conserved GLWRxC 67 motif. Boxed regions in red identify the amino acid triplets and quadruplet mutated in these experiments. (B) Pooled data (mean ± SEM) showing the effects of Ex1 mutations in γ-2 on desensitization (τ w,des ) of GluA2. Following one-way ANOVA ( F 4, 23.27 = 28.89, p = 1.01 × 10 −8 ), pairwise comparisons showed that γ-2 WT (n = 12) and all three γ-2 mutants (KGL 74–76 , KQID 78–81 , and WRT 64–66 ; n = 12, 8, and 14, respectively) slowed τ w,des compared with GluA2 alone (n = 10) ( ∗∗∗ p

    Journal: Cell Reports

    Article Title: Mapping the Interaction Sites between AMPA Receptors and TARPs Reveals a Role for the Receptor N-Terminal Domain in Channel Gating

    doi: 10.1016/j.celrep.2014.09.029

    Figure Lengend Snippet: Effects of Ex1 Mutations in TARP γ-2 (A) Sequence of the γ-2 Ex1 region surrounding the highly conserved GLWRxC 67 motif. Boxed regions in red identify the amino acid triplets and quadruplet mutated in these experiments. (B) Pooled data (mean ± SEM) showing the effects of Ex1 mutations in γ-2 on desensitization (τ w,des ) of GluA2. Following one-way ANOVA ( F 4, 23.27 = 28.89, p = 1.01 × 10 −8 ), pairwise comparisons showed that γ-2 WT (n = 12) and all three γ-2 mutants (KGL 74–76 , KQID 78–81 , and WRT 64–66 ; n = 12, 8, and 14, respectively) slowed τ w,des compared with GluA2 alone (n = 10) ( ∗∗∗ p

    Article Snippet: Membranes were blocked with 5% BSA and incubated with primary antibodies: anti-FLAG (monoclonal; Sigma-Aldrich), anti-Stargazin (polyclonal; Millipore), or anti-GluA2 (polyclonal; Alomone).

    Techniques: Sequencing

    Vezatin concentrates in spines during neuronal differentiation. A , Developmental distribution of Vezatin showing its concentration in growth cone and dendrites early during neuronal differentiation (inset, left) and in dendrites and spines in maturing and mature neurons (insets, middle and right). B , At the early stage of neuronal differentiation (left) and later, at the onset of dendritic spine development, Vezatin does not colocalize with presynaptic Syb2 (right, arrow). C , Postsynaptic Vezatin is partially codistributed with transsynaptic N-cadherin. C1 , The arrowhead and the arrow are pointing to a spine where Vezatin is (yellow) and is not (red aside green) clearly overlapping the N-cadherin signal, respectively. D , Vezatin is expressed in the soma of interneurons stained with GA D6 7 (left). Vezatin does not codistribute with GA D6 5 at the axonal compartment (right). MAP2 is used to label dendrite. E , Snapshot of a time-lapse image of a living cortical neuron transfected with Vezatin-GFP and RFP-actin showing codistribution in a hemicircumferential ring in a growth cone. Top, inset, DIV 3, fixed cultured hippocampal neuron immunostained for Vezatin and GluA2, a glutamate receptor, showing that Vezatin expression is restricted to an arc-shaped region of the growth cone. F , Vezatin colocalizes with phalloidin (F-actin) in dendrites and in developing spines at early synaptic sites (DIV 14). Scale bars, 10 μm. G , Protein expression in mouse hippocampi extracts. The anti-Vezatin antibody recognized the two major Vezatin isoforms (see text) with band intensities approximately 2× weaker in the heterozygous floxed/null ( fl/null ) compared to the floxed/floxed ( fl/fl ) extract. Other few alternatively spliced variants are expressed in minor quantities. Loading control, GAPDH.

    Journal: The Journal of Neuroscience

    Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

    doi: 10.1523/JNEUROSCI.3084-11.2012

    Figure Lengend Snippet: Vezatin concentrates in spines during neuronal differentiation. A , Developmental distribution of Vezatin showing its concentration in growth cone and dendrites early during neuronal differentiation (inset, left) and in dendrites and spines in maturing and mature neurons (insets, middle and right). B , At the early stage of neuronal differentiation (left) and later, at the onset of dendritic spine development, Vezatin does not colocalize with presynaptic Syb2 (right, arrow). C , Postsynaptic Vezatin is partially codistributed with transsynaptic N-cadherin. C1 , The arrowhead and the arrow are pointing to a spine where Vezatin is (yellow) and is not (red aside green) clearly overlapping the N-cadherin signal, respectively. D , Vezatin is expressed in the soma of interneurons stained with GA D6 7 (left). Vezatin does not codistribute with GA D6 5 at the axonal compartment (right). MAP2 is used to label dendrite. E , Snapshot of a time-lapse image of a living cortical neuron transfected with Vezatin-GFP and RFP-actin showing codistribution in a hemicircumferential ring in a growth cone. Top, inset, DIV 3, fixed cultured hippocampal neuron immunostained for Vezatin and GluA2, a glutamate receptor, showing that Vezatin expression is restricted to an arc-shaped region of the growth cone. F , Vezatin colocalizes with phalloidin (F-actin) in dendrites and in developing spines at early synaptic sites (DIV 14). Scale bars, 10 μm. G , Protein expression in mouse hippocampi extracts. The anti-Vezatin antibody recognized the two major Vezatin isoforms (see text) with band intensities approximately 2× weaker in the heterozygous floxed/null ( fl/null ) compared to the floxed/floxed ( fl/fl ) extract. Other few alternatively spliced variants are expressed in minor quantities. Loading control, GAPDH.

    Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

    Techniques: Concentration Assay, Staining, Transfection, Cell Culture, Expressing

    Subcellular spine Vezatin expression in vitro ( A – E ) and in vivo ( F – G ). A , Vezatin labels all stages of spine differentiation. Insets, Higher magnifications of a filopodium (arrowhead) and a thin spine (left). A stubby (arrow) and a mushroom-like (arrowhead) are shown on the right (top and bottom insets). B , Confocal images showing that Vezatin (green) and PSD95 (red) colocalize (yellow) at spine heads. Asterisks and arrowheads point to representative mushroom and stubby spines, respectively. Bottom right, Reconstructed view using the Imaris software package. C , Vezatin extends beyond the PSD95/PSD margin toward the neck of the spine: snapshot of a confocal spine 3D reconstruction (21 DIV). Overlay (yellow) between Vezatin (green) and PSD95 (red) staining. Both markers colocalize at the tip of the spine head, but PSD95 is more central than Vezatin. D , Vezatin is in the synaptosomal membrane fraction as PSD95 or GluA2 and GluN1 (P2). Vezatin is also in the cytosolic fraction (S2). Crude protein extract was obtained by clearing the lysate by gentle centrifugation (S1) followed by high-speed centrifugation (S2 and P2). E , High-magnification images of a 21 DIV spine whose cup-shaped base is colabeled (overlay) with Vezatin (green), phalloidin (red), and PSD95 (blue). Scale bars: A , 10 μm; B (left), E , 2 μm; B (right), 1 μm. F , Vezatin is expressed in hippocampus, cortex, and MHb. Other regions including striatum [caudate putamen (CPu) and cerebral peduncle (CP)], amygdala, and thalamus [ventral postero-medial thalamic nucleus (VPM) and ventral postero-lateral thalamic nucleus (VPL)] expressed Vezatin at a low level (objective 1.6 ×). Scale bar: 1000 μm. G , Confocal microscopy at low-power resolution indicates that Vezatin is expressed in soma (objective 20×) [SP in CA1/CA3 and interior/exterior (int/ext) in DG]. No staining is observed in the hilus (DG), except in cells attributed to astrocytes and/or inhibitory neurons. DAPI staining is used to label nuclei. G1 , Higher-power resolution (objective 40×, zoom 2.67). Left, Vezatin is in dendrites (SR in CA1). Middle, Vezatin is in spines partially codistributing with the transsynaptic N-cadherin marker (CA1 SR region). Inset, Arrowhead and arrow are pointing to spines where postsynaptic Vezatin (green) is (yellow) and is not (green aside red) overlapping N-cadherin (red). Right, Vezatin is in postsynaptic thorny excrescences (in SL in CA3) that mark the location of mossy fiber synaptic terminations (arrow). Scale bars: G , 400 μm; G1 , 100 μm. SP, stratum pyramidale; SL, stratum lacunosum.

    Journal: The Journal of Neuroscience

    Article Title: Vezatin Is Essential for Dendritic Spine Morphogenesis and Functional Synaptic Maturation

    doi: 10.1523/JNEUROSCI.3084-11.2012

    Figure Lengend Snippet: Subcellular spine Vezatin expression in vitro ( A – E ) and in vivo ( F – G ). A , Vezatin labels all stages of spine differentiation. Insets, Higher magnifications of a filopodium (arrowhead) and a thin spine (left). A stubby (arrow) and a mushroom-like (arrowhead) are shown on the right (top and bottom insets). B , Confocal images showing that Vezatin (green) and PSD95 (red) colocalize (yellow) at spine heads. Asterisks and arrowheads point to representative mushroom and stubby spines, respectively. Bottom right, Reconstructed view using the Imaris software package. C , Vezatin extends beyond the PSD95/PSD margin toward the neck of the spine: snapshot of a confocal spine 3D reconstruction (21 DIV). Overlay (yellow) between Vezatin (green) and PSD95 (red) staining. Both markers colocalize at the tip of the spine head, but PSD95 is more central than Vezatin. D , Vezatin is in the synaptosomal membrane fraction as PSD95 or GluA2 and GluN1 (P2). Vezatin is also in the cytosolic fraction (S2). Crude protein extract was obtained by clearing the lysate by gentle centrifugation (S1) followed by high-speed centrifugation (S2 and P2). E , High-magnification images of a 21 DIV spine whose cup-shaped base is colabeled (overlay) with Vezatin (green), phalloidin (red), and PSD95 (blue). Scale bars: A , 10 μm; B (left), E , 2 μm; B (right), 1 μm. F , Vezatin is expressed in hippocampus, cortex, and MHb. Other regions including striatum [caudate putamen (CPu) and cerebral peduncle (CP)], amygdala, and thalamus [ventral postero-medial thalamic nucleus (VPM) and ventral postero-lateral thalamic nucleus (VPL)] expressed Vezatin at a low level (objective 1.6 ×). Scale bar: 1000 μm. G , Confocal microscopy at low-power resolution indicates that Vezatin is expressed in soma (objective 20×) [SP in CA1/CA3 and interior/exterior (int/ext) in DG]. No staining is observed in the hilus (DG), except in cells attributed to astrocytes and/or inhibitory neurons. DAPI staining is used to label nuclei. G1 , Higher-power resolution (objective 40×, zoom 2.67). Left, Vezatin is in dendrites (SR in CA1). Middle, Vezatin is in spines partially codistributing with the transsynaptic N-cadherin marker (CA1 SR region). Inset, Arrowhead and arrow are pointing to spines where postsynaptic Vezatin (green) is (yellow) and is not (green aside red) overlapping N-cadherin (red). Right, Vezatin is in postsynaptic thorny excrescences (in SL in CA3) that mark the location of mossy fiber synaptic terminations (arrow). Scale bars: G , 400 μm; G1 , 100 μm. SP, stratum pyramidale; SL, stratum lacunosum.

    Article Snippet: Rabbit antibody anti-GluA2 was used at 1:200 (Alomone Labs).

    Techniques: Expressing, In Vitro, In Vivo, Software, Staining, Centrifugation, Confocal Microscopy, Marker

    Gliosis within the hippocampus and dentate gyrus of antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were sacrificed on day 21 post infection. Gliosis was scored as described in the Methods. The number of mice in each group is given as N above each column. Data is given as mean + SEM. †p

    Journal: Experimental neurology

    Article Title: NBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection

    doi: 10.1016/j.expneurol.2016.04.010

    Figure Lengend Snippet: Gliosis within the hippocampus and dentate gyrus of antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were sacrificed on day 21 post infection. Gliosis was scored as described in the Methods. The number of mice in each group is given as N above each column. Data is given as mean + SEM. †p

    Article Snippet: TMEV-infected mice were treated, via intraperitoneal (i.p.) injection, with MK 801 (1 mg/kg twice daily, Sigma, St. Louis, MO), GYKI-52466 (10 mg/kg twice daily, Sigma) ( ) or NBQX (approximately 22.5 mg/kg twice daily, Alomone Labs, Jerusalem, Israel) , all in a 25 μl volume, starting on day 2.5 p.i. and stopping on day 10.5 p.i.

    Techniques: Mouse Assay, Infection

    Weight change in antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were weighed daily through day 21 post infection. Data represents percent of daily weight in comparison to weight at day −1, given as mean ± standard error of the mean (SEM) for groups of 20 mice (MK 801, GYKI-52466), 19 mice (NBQX) and 60 mice (PBS). †p

    Journal: Experimental neurology

    Article Title: NBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection

    doi: 10.1016/j.expneurol.2016.04.010

    Figure Lengend Snippet: Weight change in antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were weighed daily through day 21 post infection. Data represents percent of daily weight in comparison to weight at day −1, given as mean ± standard error of the mean (SEM) for groups of 20 mice (MK 801, GYKI-52466), 19 mice (NBQX) and 60 mice (PBS). †p

    Article Snippet: TMEV-infected mice were treated, via intraperitoneal (i.p.) injection, with MK 801 (1 mg/kg twice daily, Sigma, St. Louis, MO), GYKI-52466 (10 mg/kg twice daily, Sigma) ( ) or NBQX (approximately 22.5 mg/kg twice daily, Alomone Labs, Jerusalem, Israel) , all in a 25 μl volume, starting on day 2.5 p.i. and stopping on day 10.5 p.i.

    Techniques: Mouse Assay, Infection

    Mortality in antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were monitored through day 21 post infection. Data represents percent daily survival in comparison to day 0. The numbers of mice per group at day 0 were 20 mice (MK 801, GYKI-52466), 19 mice (NBQX) and 60 mice (PBS). †p

    Journal: Experimental neurology

    Article Title: NBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection

    doi: 10.1016/j.expneurol.2016.04.010

    Figure Lengend Snippet: Mortality in antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were monitored through day 21 post infection. Data represents percent daily survival in comparison to day 0. The numbers of mice per group at day 0 were 20 mice (MK 801, GYKI-52466), 19 mice (NBQX) and 60 mice (PBS). †p

    Article Snippet: TMEV-infected mice were treated, via intraperitoneal (i.p.) injection, with MK 801 (1 mg/kg twice daily, Sigma, St. Louis, MO), GYKI-52466 (10 mg/kg twice daily, Sigma) ( ) or NBQX (approximately 22.5 mg/kg twice daily, Alomone Labs, Jerusalem, Israel) , all in a 25 μl volume, starting on day 2.5 p.i. and stopping on day 10.5 p.i.

    Techniques: Mouse Assay, Infection

    Neuronal cell loss within the hippocampus of antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were sacrificed on day 21 post infection. A. Neuronal cell loss was scored as described in the Methods. The number of mice in each group is given as N above each column. Data is given as mean + SEM. †p

    Journal: Experimental neurology

    Article Title: NBQX, a highly selective competitive antagonist of AMPA and KA ionotropic glutamate receptors, increases seizures and mortality following picornavirus infection

    doi: 10.1016/j.expneurol.2016.04.010

    Figure Lengend Snippet: Neuronal cell loss within the hippocampus of antagonist-treated mice. TMEV-infected C57BL/6J mice were treated with MK 801, GYKI-52466 and NBQX or with PBS as a control (treatment: day 2.5–10.5 post infection). Mice were sacrificed on day 21 post infection. A. Neuronal cell loss was scored as described in the Methods. The number of mice in each group is given as N above each column. Data is given as mean + SEM. †p

    Article Snippet: TMEV-infected mice were treated, via intraperitoneal (i.p.) injection, with MK 801 (1 mg/kg twice daily, Sigma, St. Louis, MO), GYKI-52466 (10 mg/kg twice daily, Sigma) ( ) or NBQX (approximately 22.5 mg/kg twice daily, Alomone Labs, Jerusalem, Israel) , all in a 25 μl volume, starting on day 2.5 p.i. and stopping on day 10.5 p.i.

    Techniques: Mouse Assay, Infection