guinea pig anti glua1  (Alomone Labs)


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    Guinea pig Anti GluR1 GluA1 extracellular Antibody
    Description:
    Guinea pig Anti GluR1 GluA1 extracellular Antibody AGP 009 is directed against an extracellular epitope the rat ionotropic glutamate receptor 1 Guinea pig Anti GluR1 GluA1 extracellular Antibody AGP 009 raised in guinea pig can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize GluR1 from human mouse and rat samples The antigen used to immunize guinea pigs is the same as Anti GluR1 GluA1 extracellular Antibody AGC 004 raised in rabbit Our line of guinea pig antibodies enables more flexibility with our products such as multiplex staining studies immunoprecipitation etc
    Catalog Number:
    AGP-009
    Price:
    397.0
    Category:
    Primary Antibody
    Applications:
    Immunocytochemistry, Immunofluorescence, Immunohistochemistry, Live Cell Imaging, Western Blot
    Purity:
    Affinity purified on immobilized antigen.
    Immunogen:
    Synthetic peptide
    Size:
    25 mcl
    Antibody Type:
    Polyclonal Primary Antibodies
    Format:
    Lyophilized Powder
    Host:
    Guinea pig
    Isotype:
    Guinea pig total IgG
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    Structured Review

    Alomone Labs guinea pig anti glua1
    Guinea pig Anti GluR1 GluA1 extracellular Antibody
    Guinea pig Anti GluR1 GluA1 extracellular Antibody AGP 009 is directed against an extracellular epitope the rat ionotropic glutamate receptor 1 Guinea pig Anti GluR1 GluA1 extracellular Antibody AGP 009 raised in guinea pig can be used in western blot immunohistochemistry and immunocytochemistry applications It has been designed to recognize GluR1 from human mouse and rat samples The antigen used to immunize guinea pigs is the same as Anti GluR1 GluA1 extracellular Antibody AGC 004 raised in rabbit Our line of guinea pig antibodies enables more flexibility with our products such as multiplex staining studies immunoprecipitation etc
    https://www.bioz.com/result/guinea pig anti glua1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    guinea pig anti glua1 - by Bioz Stars, 2021-09
    93/100 stars

    Images

    1) Product Images from "Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons"

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    Journal: Neuron

    doi: 10.1016/j.neuron.2017.10.029

    Npn-2 Selectively Associates with AMPARs in vivo and in vitro, and Forms a Complex with PlexA3 (A) Coimmunoprecipitation of FLAG-Npn-2 with different Myc tagged AMPA and NMDA receptor subunits expressed in HEK293T cells. (n = 3 experiments). (B) Coimmunoprecipitation of FLAG-Npn-2 with HA-GluA1 and HA-GluK2 from HEK293T cell lysates. (n = 3 experiments). (C) Coimmunoprecipitation of GluA1 and Npn-2 from wild type mouse brain lysates. GluA1 was immunoprecipitated from brain lysates and then immunoblotted using a Npn-2 antibody. Input was 1% of the total lysate used for the coimmunoprecipitation. Arrow indicates Npn-2 protein band (n = 4 experiments). ( D ) Coimmunoprecipitation of Npn-2 and GluA1 from wild type mouse brain lysates, and from Npn-2 −/− brain lysates as a negative control. Npn-2 was immunoprecipitated from brain lysates and immunoprecipitates immunoblotted using a GluA1 antibody. Arrow indicates GluA1 protein band. (E) Coimmunoprecipitation of HA-GluA1 with FLAG-tagged Npn-1, Npn-2, or TrkB from transfected HEK293T cells lysates. HA-GluA1 was immunoprecipitated with an HA antibody, and the resulting immunoprecipitates were subjected to immunoblotting using a FLAG antibody. (n = 3 experiments). (F) PlexA3 forms a complex with GluA1 and Npn-2. HEK293T cells were transfected with combinations of constructs expressing HA-GluA1, FLAG-Npn-2, and Myc-PlexA3. A Myc antibody was used for immunoprecipitation and the immunoprecipitates were immunoblotted using either anti-HA to detect GluA1 or anti-FLAG to detect Npn-2. (n = 3 experiments).
    Figure Legend Snippet: Npn-2 Selectively Associates with AMPARs in vivo and in vitro, and Forms a Complex with PlexA3 (A) Coimmunoprecipitation of FLAG-Npn-2 with different Myc tagged AMPA and NMDA receptor subunits expressed in HEK293T cells. (n = 3 experiments). (B) Coimmunoprecipitation of FLAG-Npn-2 with HA-GluA1 and HA-GluK2 from HEK293T cell lysates. (n = 3 experiments). (C) Coimmunoprecipitation of GluA1 and Npn-2 from wild type mouse brain lysates. GluA1 was immunoprecipitated from brain lysates and then immunoblotted using a Npn-2 antibody. Input was 1% of the total lysate used for the coimmunoprecipitation. Arrow indicates Npn-2 protein band (n = 4 experiments). ( D ) Coimmunoprecipitation of Npn-2 and GluA1 from wild type mouse brain lysates, and from Npn-2 −/− brain lysates as a negative control. Npn-2 was immunoprecipitated from brain lysates and immunoprecipitates immunoblotted using a GluA1 antibody. Arrow indicates GluA1 protein band. (E) Coimmunoprecipitation of HA-GluA1 with FLAG-tagged Npn-1, Npn-2, or TrkB from transfected HEK293T cells lysates. HA-GluA1 was immunoprecipitated with an HA antibody, and the resulting immunoprecipitates were subjected to immunoblotting using a FLAG antibody. (n = 3 experiments). (F) PlexA3 forms a complex with GluA1 and Npn-2. HEK293T cells were transfected with combinations of constructs expressing HA-GluA1, FLAG-Npn-2, and Myc-PlexA3. A Myc antibody was used for immunoprecipitation and the immunoprecipitates were immunoblotted using either anti-HA to detect GluA1 or anti-FLAG to detect Npn-2. (n = 3 experiments).

    Techniques Used: In Vivo, In Vitro, Immunoprecipitation, Negative Control, Transfection, Construct, Expressing

    Npn-2 Associates with GluA1 Through Both CUB Domains and is Required Cell-autonomously for Bicuculline-induced GluA1 Downscaling (A) Schematic diagrams of FLAG-Npn-2 proteins with CUB1, CUB2, CUB1 and CUB2, Fv, or MAM domain deletions used in (B). Dash marks indicate domain(s) that have been deleted from the full-length protein. (B) Coimmunoprecipitation of FLAG-Npn-2 proteins harboring the deletions shown in (A) with GluA1 from transfected HEK293T cell lysates. An HA antibody was used to immunoprecipitate HA-GluA1. Deletion of either Npn-2 CUB domain results in failure of Npn-2 binding to GluA1 (n = 3 experiments). (C) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-1 or FLAG-Npn-1 Npn-2CUB (a Npn-1 chimeric protein containing Npn-2 CUB domains in place of Npn-1 CUB domains) in transfected HEK293T cell lysates. GluA1 binds to Npn-1 Npn-2CUB but not to Npn-1 (n = 3 experiments). (D) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-2 or FLAG-Npn-2 Npn-1CUB2 (a Npn-2/1 chimeric protein containing the Npn-1 CUB domain 2 in place of the Npn-2 CUB domain 2) in transfected HEK293T cell lysates. (n = 3 experiments). (E) Cell surface GluA1 immunostaining following bicuculline or TTX treatment of Npn-2 F /− cortical cultures (14 DIV) transfected with constructs expressing various Npn-2 proteins. pCAGGS-Cre-IRES-GFP was used to remove Npn-2 in individual neurons, and plasmids expressing Npn-2, Npn-2 ΔCUB2 , Npn-2 Sema3F− , or Npn-2 Npn-1CUB2 (a Npn-2 chimeric protein containing the Npn-1 CUB2 domain in place of the Npn-2 CUB2 domain) were transfected along with the Cre-expressing plasmids (the pCAGGS-IRES-GFP was used as a control). Transfected neurons were identified by GFP fluorescence. Shown are segments of mouse cortical neuron dendrites immunostained for GluA1 and GFP. (F) Quantification of cell surface GluA1 immunostaining in transfected neurons in (E) (n = 45 transfected neurons for each condition, and 10–15 dendritic segments from each individual neuron were assessed). *P
    Figure Legend Snippet: Npn-2 Associates with GluA1 Through Both CUB Domains and is Required Cell-autonomously for Bicuculline-induced GluA1 Downscaling (A) Schematic diagrams of FLAG-Npn-2 proteins with CUB1, CUB2, CUB1 and CUB2, Fv, or MAM domain deletions used in (B). Dash marks indicate domain(s) that have been deleted from the full-length protein. (B) Coimmunoprecipitation of FLAG-Npn-2 proteins harboring the deletions shown in (A) with GluA1 from transfected HEK293T cell lysates. An HA antibody was used to immunoprecipitate HA-GluA1. Deletion of either Npn-2 CUB domain results in failure of Npn-2 binding to GluA1 (n = 3 experiments). (C) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-1 or FLAG-Npn-1 Npn-2CUB (a Npn-1 chimeric protein containing Npn-2 CUB domains in place of Npn-1 CUB domains) in transfected HEK293T cell lysates. GluA1 binds to Npn-1 Npn-2CUB but not to Npn-1 (n = 3 experiments). (D) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-2 or FLAG-Npn-2 Npn-1CUB2 (a Npn-2/1 chimeric protein containing the Npn-1 CUB domain 2 in place of the Npn-2 CUB domain 2) in transfected HEK293T cell lysates. (n = 3 experiments). (E) Cell surface GluA1 immunostaining following bicuculline or TTX treatment of Npn-2 F /− cortical cultures (14 DIV) transfected with constructs expressing various Npn-2 proteins. pCAGGS-Cre-IRES-GFP was used to remove Npn-2 in individual neurons, and plasmids expressing Npn-2, Npn-2 ΔCUB2 , Npn-2 Sema3F− , or Npn-2 Npn-1CUB2 (a Npn-2 chimeric protein containing the Npn-1 CUB2 domain in place of the Npn-2 CUB2 domain) were transfected along with the Cre-expressing plasmids (the pCAGGS-IRES-GFP was used as a control). Transfected neurons were identified by GFP fluorescence. Shown are segments of mouse cortical neuron dendrites immunostained for GluA1 and GFP. (F) Quantification of cell surface GluA1 immunostaining in transfected neurons in (E) (n = 45 transfected neurons for each condition, and 10–15 dendritic segments from each individual neuron were assessed). *P

    Techniques Used: Transfection, Binding Assay, Immunostaining, Construct, Expressing, Fluorescence

    Sema3F −/− Cortical Neurons in Culture do not Exhibit Bicuculline-induced Downscaling of Cell Surface AMPA Receptors (A) Cell surface GluA1 immunostaining of wild type and Sema3F −/− cortical cultures (14 DIV) treated for 48 hrs with TTX, bicuculline or control media. (B) Quantification of cell surface GluA1 in wild type and Sema3F −/− cortical cultures relative to controls following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR Methods). (C) Cortical neurons (14 DIV) from wild type and Sema3F −/− cortical cultures were treated with bicuculline, TTX or control media for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 and GluA2 expression. (D) Quantification of cell surface GluA1 and GluA2 levels in wild type and Sema3F −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). (E) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM) for indicated durations, followed by cell surface biotinylation and Western blotting for cell surface GluA1. (F) Quantification of cell surface GluA1 levels in Sema3F-treated cortical cultures at different time points (n = 3 experiments). (G) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM), bicuculline, or Sema3F plus bicuculline for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 expression (n = 3 experiments). (H) Quantification of (G). * P
    Figure Legend Snippet: Sema3F −/− Cortical Neurons in Culture do not Exhibit Bicuculline-induced Downscaling of Cell Surface AMPA Receptors (A) Cell surface GluA1 immunostaining of wild type and Sema3F −/− cortical cultures (14 DIV) treated for 48 hrs with TTX, bicuculline or control media. (B) Quantification of cell surface GluA1 in wild type and Sema3F −/− cortical cultures relative to controls following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR Methods). (C) Cortical neurons (14 DIV) from wild type and Sema3F −/− cortical cultures were treated with bicuculline, TTX or control media for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 and GluA2 expression. (D) Quantification of cell surface GluA1 and GluA2 levels in wild type and Sema3F −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). (E) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM) for indicated durations, followed by cell surface biotinylation and Western blotting for cell surface GluA1. (F) Quantification of cell surface GluA1 levels in Sema3F-treated cortical cultures at different time points (n = 3 experiments). (G) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM), bicuculline, or Sema3F plus bicuculline for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 expression (n = 3 experiments). (H) Quantification of (G). * P

    Techniques Used: Immunostaining, Western Blot, Expressing

    Bicuculline-dependent Cell Surface AMPA Receptor Downscaling is Abrogated in Npn-2 Mutant Cortical Neurons (A) Cultured cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were transfected with GFP at DIV 12 and stained with a GFP antibody to reveal dendritic spines. An HA antibody was used to detect expression of endogenous HA Npn-2. Lower panels, enlarged area from upper panels as indicated. Arrows, examples of dendritic spines that include HA Npn-2 puncta (n = 3 independent cultures, 28 neurons). (B) Cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were co-stained with anti-GluA1 and anti-HA. Lower panels, enlarged area from upper panels as indicated. Arrow, examples of colocalization of GluA1 and HA Npn-2 + puncta (n = 3 independent cultures, 31 neurons; see STAR Methods). (C) Cell surface GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E14.5, 14 DIV) treated for 48 additional hrs with TTX, bicuculline, or control media. (D) Quantification of cell surface GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR methods). (E) Wild type and Npn-2 −/− cortical neurons (14 DIV) were treated with bicuculline, TTX, or control media for 48 hrs, followed by cell surface biotinylation and Western blotting to reveal GluA1 and GluA2 cell surface expression. (F) Quantification of cell surface GluA1 and GluA2 receptor levels in wild type and Npn-2 −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). *P
    Figure Legend Snippet: Bicuculline-dependent Cell Surface AMPA Receptor Downscaling is Abrogated in Npn-2 Mutant Cortical Neurons (A) Cultured cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were transfected with GFP at DIV 12 and stained with a GFP antibody to reveal dendritic spines. An HA antibody was used to detect expression of endogenous HA Npn-2. Lower panels, enlarged area from upper panels as indicated. Arrows, examples of dendritic spines that include HA Npn-2 puncta (n = 3 independent cultures, 28 neurons). (B) Cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were co-stained with anti-GluA1 and anti-HA. Lower panels, enlarged area from upper panels as indicated. Arrow, examples of colocalization of GluA1 and HA Npn-2 + puncta (n = 3 independent cultures, 31 neurons; see STAR Methods). (C) Cell surface GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E14.5, 14 DIV) treated for 48 additional hrs with TTX, bicuculline, or control media. (D) Quantification of cell surface GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR methods). (E) Wild type and Npn-2 −/− cortical neurons (14 DIV) were treated with bicuculline, TTX, or control media for 48 hrs, followed by cell surface biotinylation and Western blotting to reveal GluA1 and GluA2 cell surface expression. (F) Quantification of cell surface GluA1 and GluA2 receptor levels in wild type and Npn-2 −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). *P

    Techniques Used: Mutagenesis, Cell Culture, Derivative Assay, Transfection, Staining, Expressing, Immunostaining, Western Blot

    Npn-2 −/− Cortical Neurons in Culture Show Abrogated Bicuculline-induced Synaptic Downscaling (A) Whole cell recordings were performed on wild type or Npn-2 −/− cortical neurons (E18, 14 DIV) with or without 48 hrs of bicuculline treatment. Representative traces of spontaneous AMPA receptor-mediated mEPSCs recorded from wild type and Npn-2 −/− neurons. (B) Quantification of mEPSC amplitudes from wild type neurons (Control= 15.11 ± 0.67 pA, n = 19 neurons, and Bicuculline= 12.98 ± 0.34 pA, n = 24 neurons; p = 0.0266, two-way ANOVA with Sidak’s multiple comparison test ). Quantification of mEPSC amplitude from Npn-2 −/− neurons in culture (Control= 14.09 ± 0.85 pA, n = 16 neurons, and Bicuculline= 14.89 ± 0.65 pA, n = 20 neurons; p=0.6244; wild type Bicuculline and Npn-2 −/− Bicuculline, P = 0.04; two-way ANOVA with Sidak’s multiple comparison test, two-way ANOVA interaction term: P = 0.0213). (C) Surface synaptic GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E18, 14 DIV) treated for 48 additional hours with bicuculline or control media. Synaptic GluA1 was identified by co-staining with a PSD95 antibody. (D) Quantification of synaptic GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following bicuculline treatments (n = 35–40 neurons for each condition; see STAR Methods). *P
    Figure Legend Snippet: Npn-2 −/− Cortical Neurons in Culture Show Abrogated Bicuculline-induced Synaptic Downscaling (A) Whole cell recordings were performed on wild type or Npn-2 −/− cortical neurons (E18, 14 DIV) with or without 48 hrs of bicuculline treatment. Representative traces of spontaneous AMPA receptor-mediated mEPSCs recorded from wild type and Npn-2 −/− neurons. (B) Quantification of mEPSC amplitudes from wild type neurons (Control= 15.11 ± 0.67 pA, n = 19 neurons, and Bicuculline= 12.98 ± 0.34 pA, n = 24 neurons; p = 0.0266, two-way ANOVA with Sidak’s multiple comparison test ). Quantification of mEPSC amplitude from Npn-2 −/− neurons in culture (Control= 14.09 ± 0.85 pA, n = 16 neurons, and Bicuculline= 14.89 ± 0.65 pA, n = 20 neurons; p=0.6244; wild type Bicuculline and Npn-2 −/− Bicuculline, P = 0.04; two-way ANOVA with Sidak’s multiple comparison test, two-way ANOVA interaction term: P = 0.0213). (C) Surface synaptic GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E18, 14 DIV) treated for 48 additional hours with bicuculline or control media. Synaptic GluA1 was identified by co-staining with a PSD95 antibody. (D) Quantification of synaptic GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following bicuculline treatments (n = 35–40 neurons for each condition; see STAR Methods). *P

    Techniques Used: Immunostaining, Staining

    Sema3F and Neuronal Activity Regulate the Interaction between Npn-2 and GluA1 (A) Regulation of the interaction between HA-GluA1 and FLAG-Npn-2 in HEK293T cells by Sema3F. HEK293T cells transfected with HA-GluA1, FLAG-Npn-2, and Myc-PlexA3 constructs were treated with AP-Sema3F (5 nM) for the indicated times. Npn-2 was co-immunoprecipitated with GluA1 from the transfected cell lysates using an HA antibody. (B) Quantification of Npn-2 coimmunoprecipitations with GluA1 from transfected HEK293T cell lysates following Sema3F treatment. (n = 3 experiments). For quantification, the intensity of coimmunoprecipitated Npn-2 was normalized to the intensity of Npn-2 input, and the value of the normalized control sample (0 min) was set as 100%. (C) Sema3F regulation of the interaction between GluA1 and Npn-2 in cortical neurons. 14 DIV cortical neurons were treated with 5 nM Sema3F for the indicated times, and cell lysates were collected and subjected to coimmunoprecipitation using a GluA1 antibody. (D) Quantification of the Npn-2 interaction with GluA1 upon Sema3F treatment relative to the untreated control, presented in C (n=3 experiments). Quantification was performed as described in B. (E) HEK293T cells were transfected with constructs expressing either wild type FLAG-Npn-2 or FLAG-Npn-2 Sema3F− together with HA-GluA1 and Myc-PlexA3. Transfected cells were treated with either AP or AP-Sema3F for 30 min. (F) Quantification of experiments in E, showing that Sema3F treatment fails to modulate the interaction between Npn-2 Sema3F− (Npn-2 lacking the ability to bind Sema3F) and GluA1 (n = 3 experiments). Quantification was performed as described in B. (G) Neuronal activity regulates the interaction between Npn-2 and GluA1. Cortical cultures (14 DIV) derived from wild type or Sema3F −/− embryos were treated with bicuculline or control media for 48 hrs; cell lysates were collected and subjected to co-immunoprecipitation with a GluA1 antibody. (H) Quantification of G. Coimmunoprecipitated Npn-2 following bicuculline treatment was quantified relative to the untreated sample. Quantification was performed as described in B (n = 3 experiments). *P
    Figure Legend Snippet: Sema3F and Neuronal Activity Regulate the Interaction between Npn-2 and GluA1 (A) Regulation of the interaction between HA-GluA1 and FLAG-Npn-2 in HEK293T cells by Sema3F. HEK293T cells transfected with HA-GluA1, FLAG-Npn-2, and Myc-PlexA3 constructs were treated with AP-Sema3F (5 nM) for the indicated times. Npn-2 was co-immunoprecipitated with GluA1 from the transfected cell lysates using an HA antibody. (B) Quantification of Npn-2 coimmunoprecipitations with GluA1 from transfected HEK293T cell lysates following Sema3F treatment. (n = 3 experiments). For quantification, the intensity of coimmunoprecipitated Npn-2 was normalized to the intensity of Npn-2 input, and the value of the normalized control sample (0 min) was set as 100%. (C) Sema3F regulation of the interaction between GluA1 and Npn-2 in cortical neurons. 14 DIV cortical neurons were treated with 5 nM Sema3F for the indicated times, and cell lysates were collected and subjected to coimmunoprecipitation using a GluA1 antibody. (D) Quantification of the Npn-2 interaction with GluA1 upon Sema3F treatment relative to the untreated control, presented in C (n=3 experiments). Quantification was performed as described in B. (E) HEK293T cells were transfected with constructs expressing either wild type FLAG-Npn-2 or FLAG-Npn-2 Sema3F− together with HA-GluA1 and Myc-PlexA3. Transfected cells were treated with either AP or AP-Sema3F for 30 min. (F) Quantification of experiments in E, showing that Sema3F treatment fails to modulate the interaction between Npn-2 Sema3F− (Npn-2 lacking the ability to bind Sema3F) and GluA1 (n = 3 experiments). Quantification was performed as described in B. (G) Neuronal activity regulates the interaction between Npn-2 and GluA1. Cortical cultures (14 DIV) derived from wild type or Sema3F −/− embryos were treated with bicuculline or control media for 48 hrs; cell lysates were collected and subjected to co-immunoprecipitation with a GluA1 antibody. (H) Quantification of G. Coimmunoprecipitated Npn-2 following bicuculline treatment was quantified relative to the untreated sample. Quantification was performed as described in B (n = 3 experiments). *P

    Techniques Used: Activity Assay, Transfection, Construct, Immunoprecipitation, Expressing, Derivative Assay

    2) Product Images from "Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes"

    Article Title: Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes

    Journal: Scientific Reports

    doi: 10.1038/srep44817

    Immunostaining of AMPA receptor subunits in the olfactory bulb. ( a ) GluA2 immunoreactivity (green) was detected in the external plexiform layer (EPL) and in cell bodies surrounding glomeruli. Glomeruli are indicated by asterisks. Moderate GluA2 immunoreactivity was also found in astrocytes highlighted by GFAP immunoreactivity (red), as indicated by yellow pixels in the merged image. Arrows point to astrocyte structures that were colabeled with GluA immunoreactivity. Nuclei were stained with Hoechst 33342 (blue). ( b ) GluA1 and GFAP colocalization. ( c ) GluA4 and GFAP colocalization. Scale bars: 20 μm.
    Figure Legend Snippet: Immunostaining of AMPA receptor subunits in the olfactory bulb. ( a ) GluA2 immunoreactivity (green) was detected in the external plexiform layer (EPL) and in cell bodies surrounding glomeruli. Glomeruli are indicated by asterisks. Moderate GluA2 immunoreactivity was also found in astrocytes highlighted by GFAP immunoreactivity (red), as indicated by yellow pixels in the merged image. Arrows point to astrocyte structures that were colabeled with GluA immunoreactivity. Nuclei were stained with Hoechst 33342 (blue). ( b ) GluA1 and GFAP colocalization. ( c ) GluA4 and GFAP colocalization. Scale bars: 20 μm.

    Techniques Used: Immunostaining, Staining

    Related Articles

    Incubation:

    Article Title: Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes
    Article Snippet: .. Slices were cut with a vibratome (VT1000, Leica) and incubated with the primary antibodies anti-GluA1 (guinea pig; 1:200; Alomone Labs), anti-GluA2 (rabbit; 1:200; Millipore, Darmstadt, Germany), anti-GluA4 (rabbit; 1:200; Millipore), anti-GFAP (rabbit, 1:000, Dako, Hamburg, Germany), anti-GFAP (chicken; 1:500; Abcam) and anti-GFP (chicken, 1:500, SySy, Göttingen, Germany). ..

    Article Title: Seizure-related 6 homolog like 2 autoimmunity
    Article Snippet: .. Anti-GluA1 (1:200, AGP-009, Alomone Labs, Jerusalem, Israel) was incubated in addition to serum of patient 4, as previously described. ..

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    Alomone Labs guinea pig anti glua1
    Npn-2 Selectively Associates with AMPARs in vivo and in vitro, and Forms a Complex with PlexA3 (A) Coimmunoprecipitation of FLAG-Npn-2 with different Myc tagged AMPA and NMDA receptor subunits expressed in HEK293T cells. (n = 3 experiments). (B) Coimmunoprecipitation of FLAG-Npn-2 with <t>HA-GluA1</t> and HA-GluK2 from HEK293T cell lysates. (n = 3 experiments). (C) Coimmunoprecipitation of GluA1 and Npn-2 from wild type mouse brain lysates. GluA1 was immunoprecipitated from brain lysates and then immunoblotted using a Npn-2 antibody. Input was 1% of the total lysate used for the coimmunoprecipitation. Arrow indicates Npn-2 protein band (n = 4 experiments). ( D ) Coimmunoprecipitation of Npn-2 and GluA1 from wild type mouse brain lysates, and from Npn-2 −/− brain lysates as a negative control. Npn-2 was immunoprecipitated from brain lysates and immunoprecipitates immunoblotted using a GluA1 antibody. Arrow indicates GluA1 protein band. (E) Coimmunoprecipitation of HA-GluA1 with FLAG-tagged Npn-1, Npn-2, or TrkB from transfected HEK293T cells lysates. HA-GluA1 was immunoprecipitated with an HA antibody, and the resulting immunoprecipitates were subjected to immunoblotting using a FLAG antibody. (n = 3 experiments). (F) PlexA3 forms a complex with GluA1 and Npn-2. HEK293T cells were transfected with combinations of constructs expressing HA-GluA1, FLAG-Npn-2, and Myc-PlexA3. A Myc antibody was used for immunoprecipitation and the immunoprecipitates were immunoblotted using either anti-HA to detect GluA1 or anti-FLAG to detect Npn-2. (n = 3 experiments).
    Guinea Pig Anti Glua1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/guinea pig anti glua1/product/Alomone Labs
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    Npn-2 Selectively Associates with AMPARs in vivo and in vitro, and Forms a Complex with PlexA3 (A) Coimmunoprecipitation of FLAG-Npn-2 with different Myc tagged AMPA and NMDA receptor subunits expressed in HEK293T cells. (n = 3 experiments). (B) Coimmunoprecipitation of FLAG-Npn-2 with HA-GluA1 and HA-GluK2 from HEK293T cell lysates. (n = 3 experiments). (C) Coimmunoprecipitation of GluA1 and Npn-2 from wild type mouse brain lysates. GluA1 was immunoprecipitated from brain lysates and then immunoblotted using a Npn-2 antibody. Input was 1% of the total lysate used for the coimmunoprecipitation. Arrow indicates Npn-2 protein band (n = 4 experiments). ( D ) Coimmunoprecipitation of Npn-2 and GluA1 from wild type mouse brain lysates, and from Npn-2 −/− brain lysates as a negative control. Npn-2 was immunoprecipitated from brain lysates and immunoprecipitates immunoblotted using a GluA1 antibody. Arrow indicates GluA1 protein band. (E) Coimmunoprecipitation of HA-GluA1 with FLAG-tagged Npn-1, Npn-2, or TrkB from transfected HEK293T cells lysates. HA-GluA1 was immunoprecipitated with an HA antibody, and the resulting immunoprecipitates were subjected to immunoblotting using a FLAG antibody. (n = 3 experiments). (F) PlexA3 forms a complex with GluA1 and Npn-2. HEK293T cells were transfected with combinations of constructs expressing HA-GluA1, FLAG-Npn-2, and Myc-PlexA3. A Myc antibody was used for immunoprecipitation and the immunoprecipitates were immunoblotted using either anti-HA to detect GluA1 or anti-FLAG to detect Npn-2. (n = 3 experiments).

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Npn-2 Selectively Associates with AMPARs in vivo and in vitro, and Forms a Complex with PlexA3 (A) Coimmunoprecipitation of FLAG-Npn-2 with different Myc tagged AMPA and NMDA receptor subunits expressed in HEK293T cells. (n = 3 experiments). (B) Coimmunoprecipitation of FLAG-Npn-2 with HA-GluA1 and HA-GluK2 from HEK293T cell lysates. (n = 3 experiments). (C) Coimmunoprecipitation of GluA1 and Npn-2 from wild type mouse brain lysates. GluA1 was immunoprecipitated from brain lysates and then immunoblotted using a Npn-2 antibody. Input was 1% of the total lysate used for the coimmunoprecipitation. Arrow indicates Npn-2 protein band (n = 4 experiments). ( D ) Coimmunoprecipitation of Npn-2 and GluA1 from wild type mouse brain lysates, and from Npn-2 −/− brain lysates as a negative control. Npn-2 was immunoprecipitated from brain lysates and immunoprecipitates immunoblotted using a GluA1 antibody. Arrow indicates GluA1 protein band. (E) Coimmunoprecipitation of HA-GluA1 with FLAG-tagged Npn-1, Npn-2, or TrkB from transfected HEK293T cells lysates. HA-GluA1 was immunoprecipitated with an HA antibody, and the resulting immunoprecipitates were subjected to immunoblotting using a FLAG antibody. (n = 3 experiments). (F) PlexA3 forms a complex with GluA1 and Npn-2. HEK293T cells were transfected with combinations of constructs expressing HA-GluA1, FLAG-Npn-2, and Myc-PlexA3. A Myc antibody was used for immunoprecipitation and the immunoprecipitates were immunoblotted using either anti-HA to detect GluA1 or anti-FLAG to detect Npn-2. (n = 3 experiments).

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: In Vivo, In Vitro, Immunoprecipitation, Negative Control, Transfection, Construct, Expressing

    Npn-2 Associates with GluA1 Through Both CUB Domains and is Required Cell-autonomously for Bicuculline-induced GluA1 Downscaling (A) Schematic diagrams of FLAG-Npn-2 proteins with CUB1, CUB2, CUB1 and CUB2, Fv, or MAM domain deletions used in (B). Dash marks indicate domain(s) that have been deleted from the full-length protein. (B) Coimmunoprecipitation of FLAG-Npn-2 proteins harboring the deletions shown in (A) with GluA1 from transfected HEK293T cell lysates. An HA antibody was used to immunoprecipitate HA-GluA1. Deletion of either Npn-2 CUB domain results in failure of Npn-2 binding to GluA1 (n = 3 experiments). (C) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-1 or FLAG-Npn-1 Npn-2CUB (a Npn-1 chimeric protein containing Npn-2 CUB domains in place of Npn-1 CUB domains) in transfected HEK293T cell lysates. GluA1 binds to Npn-1 Npn-2CUB but not to Npn-1 (n = 3 experiments). (D) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-2 or FLAG-Npn-2 Npn-1CUB2 (a Npn-2/1 chimeric protein containing the Npn-1 CUB domain 2 in place of the Npn-2 CUB domain 2) in transfected HEK293T cell lysates. (n = 3 experiments). (E) Cell surface GluA1 immunostaining following bicuculline or TTX treatment of Npn-2 F /− cortical cultures (14 DIV) transfected with constructs expressing various Npn-2 proteins. pCAGGS-Cre-IRES-GFP was used to remove Npn-2 in individual neurons, and plasmids expressing Npn-2, Npn-2 ΔCUB2 , Npn-2 Sema3F− , or Npn-2 Npn-1CUB2 (a Npn-2 chimeric protein containing the Npn-1 CUB2 domain in place of the Npn-2 CUB2 domain) were transfected along with the Cre-expressing plasmids (the pCAGGS-IRES-GFP was used as a control). Transfected neurons were identified by GFP fluorescence. Shown are segments of mouse cortical neuron dendrites immunostained for GluA1 and GFP. (F) Quantification of cell surface GluA1 immunostaining in transfected neurons in (E) (n = 45 transfected neurons for each condition, and 10–15 dendritic segments from each individual neuron were assessed). *P

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Npn-2 Associates with GluA1 Through Both CUB Domains and is Required Cell-autonomously for Bicuculline-induced GluA1 Downscaling (A) Schematic diagrams of FLAG-Npn-2 proteins with CUB1, CUB2, CUB1 and CUB2, Fv, or MAM domain deletions used in (B). Dash marks indicate domain(s) that have been deleted from the full-length protein. (B) Coimmunoprecipitation of FLAG-Npn-2 proteins harboring the deletions shown in (A) with GluA1 from transfected HEK293T cell lysates. An HA antibody was used to immunoprecipitate HA-GluA1. Deletion of either Npn-2 CUB domain results in failure of Npn-2 binding to GluA1 (n = 3 experiments). (C) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-1 or FLAG-Npn-1 Npn-2CUB (a Npn-1 chimeric protein containing Npn-2 CUB domains in place of Npn-1 CUB domains) in transfected HEK293T cell lysates. GluA1 binds to Npn-1 Npn-2CUB but not to Npn-1 (n = 3 experiments). (D) Coimmunoprecipitation of HA-GluA1 with FLAG-Npn-2 or FLAG-Npn-2 Npn-1CUB2 (a Npn-2/1 chimeric protein containing the Npn-1 CUB domain 2 in place of the Npn-2 CUB domain 2) in transfected HEK293T cell lysates. (n = 3 experiments). (E) Cell surface GluA1 immunostaining following bicuculline or TTX treatment of Npn-2 F /− cortical cultures (14 DIV) transfected with constructs expressing various Npn-2 proteins. pCAGGS-Cre-IRES-GFP was used to remove Npn-2 in individual neurons, and plasmids expressing Npn-2, Npn-2 ΔCUB2 , Npn-2 Sema3F− , or Npn-2 Npn-1CUB2 (a Npn-2 chimeric protein containing the Npn-1 CUB2 domain in place of the Npn-2 CUB2 domain) were transfected along with the Cre-expressing plasmids (the pCAGGS-IRES-GFP was used as a control). Transfected neurons were identified by GFP fluorescence. Shown are segments of mouse cortical neuron dendrites immunostained for GluA1 and GFP. (F) Quantification of cell surface GluA1 immunostaining in transfected neurons in (E) (n = 45 transfected neurons for each condition, and 10–15 dendritic segments from each individual neuron were assessed). *P

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: Transfection, Binding Assay, Immunostaining, Construct, Expressing, Fluorescence

    Sema3F −/− Cortical Neurons in Culture do not Exhibit Bicuculline-induced Downscaling of Cell Surface AMPA Receptors (A) Cell surface GluA1 immunostaining of wild type and Sema3F −/− cortical cultures (14 DIV) treated for 48 hrs with TTX, bicuculline or control media. (B) Quantification of cell surface GluA1 in wild type and Sema3F −/− cortical cultures relative to controls following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR Methods). (C) Cortical neurons (14 DIV) from wild type and Sema3F −/− cortical cultures were treated with bicuculline, TTX or control media for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 and GluA2 expression. (D) Quantification of cell surface GluA1 and GluA2 levels in wild type and Sema3F −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). (E) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM) for indicated durations, followed by cell surface biotinylation and Western blotting for cell surface GluA1. (F) Quantification of cell surface GluA1 levels in Sema3F-treated cortical cultures at different time points (n = 3 experiments). (G) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM), bicuculline, or Sema3F plus bicuculline for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 expression (n = 3 experiments). (H) Quantification of (G). * P

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Sema3F −/− Cortical Neurons in Culture do not Exhibit Bicuculline-induced Downscaling of Cell Surface AMPA Receptors (A) Cell surface GluA1 immunostaining of wild type and Sema3F −/− cortical cultures (14 DIV) treated for 48 hrs with TTX, bicuculline or control media. (B) Quantification of cell surface GluA1 in wild type and Sema3F −/− cortical cultures relative to controls following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR Methods). (C) Cortical neurons (14 DIV) from wild type and Sema3F −/− cortical cultures were treated with bicuculline, TTX or control media for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 and GluA2 expression. (D) Quantification of cell surface GluA1 and GluA2 levels in wild type and Sema3F −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). (E) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM) for indicated durations, followed by cell surface biotinylation and Western blotting for cell surface GluA1. (F) Quantification of cell surface GluA1 levels in Sema3F-treated cortical cultures at different time points (n = 3 experiments). (G) Cortical neurons from wild type cortical cultures were treated with Sema3F (5 nM), bicuculline, or Sema3F plus bicuculline for 48 hrs followed by cell surface biotinylation and Western blotting to reveal cell surface GluA1 expression (n = 3 experiments). (H) Quantification of (G). * P

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: Immunostaining, Western Blot, Expressing

    Bicuculline-dependent Cell Surface AMPA Receptor Downscaling is Abrogated in Npn-2 Mutant Cortical Neurons (A) Cultured cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were transfected with GFP at DIV 12 and stained with a GFP antibody to reveal dendritic spines. An HA antibody was used to detect expression of endogenous HA Npn-2. Lower panels, enlarged area from upper panels as indicated. Arrows, examples of dendritic spines that include HA Npn-2 puncta (n = 3 independent cultures, 28 neurons). (B) Cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were co-stained with anti-GluA1 and anti-HA. Lower panels, enlarged area from upper panels as indicated. Arrow, examples of colocalization of GluA1 and HA Npn-2 + puncta (n = 3 independent cultures, 31 neurons; see STAR Methods). (C) Cell surface GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E14.5, 14 DIV) treated for 48 additional hrs with TTX, bicuculline, or control media. (D) Quantification of cell surface GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR methods). (E) Wild type and Npn-2 −/− cortical neurons (14 DIV) were treated with bicuculline, TTX, or control media for 48 hrs, followed by cell surface biotinylation and Western blotting to reveal GluA1 and GluA2 cell surface expression. (F) Quantification of cell surface GluA1 and GluA2 receptor levels in wild type and Npn-2 −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). *P

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Bicuculline-dependent Cell Surface AMPA Receptor Downscaling is Abrogated in Npn-2 Mutant Cortical Neurons (A) Cultured cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were transfected with GFP at DIV 12 and stained with a GFP antibody to reveal dendritic spines. An HA antibody was used to detect expression of endogenous HA Npn-2. Lower panels, enlarged area from upper panels as indicated. Arrows, examples of dendritic spines that include HA Npn-2 puncta (n = 3 independent cultures, 28 neurons). (B) Cortical neurons (17 DIV) derived from E18 HA Npn-2 embryos were co-stained with anti-GluA1 and anti-HA. Lower panels, enlarged area from upper panels as indicated. Arrow, examples of colocalization of GluA1 and HA Npn-2 + puncta (n = 3 independent cultures, 31 neurons; see STAR Methods). (C) Cell surface GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E14.5, 14 DIV) treated for 48 additional hrs with TTX, bicuculline, or control media. (D) Quantification of cell surface GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following TTX or bicuculline treatments (n = 50 neurons for each condition; see STAR methods). (E) Wild type and Npn-2 −/− cortical neurons (14 DIV) were treated with bicuculline, TTX, or control media for 48 hrs, followed by cell surface biotinylation and Western blotting to reveal GluA1 and GluA2 cell surface expression. (F) Quantification of cell surface GluA1 and GluA2 receptor levels in wild type and Npn-2 −/− cortical cultures treated with TTX or bicuculline relative to control cultures (n = 5 experiments). *P

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: Mutagenesis, Cell Culture, Derivative Assay, Transfection, Staining, Expressing, Immunostaining, Western Blot

    Npn-2 −/− Cortical Neurons in Culture Show Abrogated Bicuculline-induced Synaptic Downscaling (A) Whole cell recordings were performed on wild type or Npn-2 −/− cortical neurons (E18, 14 DIV) with or without 48 hrs of bicuculline treatment. Representative traces of spontaneous AMPA receptor-mediated mEPSCs recorded from wild type and Npn-2 −/− neurons. (B) Quantification of mEPSC amplitudes from wild type neurons (Control= 15.11 ± 0.67 pA, n = 19 neurons, and Bicuculline= 12.98 ± 0.34 pA, n = 24 neurons; p = 0.0266, two-way ANOVA with Sidak’s multiple comparison test ). Quantification of mEPSC amplitude from Npn-2 −/− neurons in culture (Control= 14.09 ± 0.85 pA, n = 16 neurons, and Bicuculline= 14.89 ± 0.65 pA, n = 20 neurons; p=0.6244; wild type Bicuculline and Npn-2 −/− Bicuculline, P = 0.04; two-way ANOVA with Sidak’s multiple comparison test, two-way ANOVA interaction term: P = 0.0213). (C) Surface synaptic GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E18, 14 DIV) treated for 48 additional hours with bicuculline or control media. Synaptic GluA1 was identified by co-staining with a PSD95 antibody. (D) Quantification of synaptic GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following bicuculline treatments (n = 35–40 neurons for each condition; see STAR Methods). *P

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Npn-2 −/− Cortical Neurons in Culture Show Abrogated Bicuculline-induced Synaptic Downscaling (A) Whole cell recordings were performed on wild type or Npn-2 −/− cortical neurons (E18, 14 DIV) with or without 48 hrs of bicuculline treatment. Representative traces of spontaneous AMPA receptor-mediated mEPSCs recorded from wild type and Npn-2 −/− neurons. (B) Quantification of mEPSC amplitudes from wild type neurons (Control= 15.11 ± 0.67 pA, n = 19 neurons, and Bicuculline= 12.98 ± 0.34 pA, n = 24 neurons; p = 0.0266, two-way ANOVA with Sidak’s multiple comparison test ). Quantification of mEPSC amplitude from Npn-2 −/− neurons in culture (Control= 14.09 ± 0.85 pA, n = 16 neurons, and Bicuculline= 14.89 ± 0.65 pA, n = 20 neurons; p=0.6244; wild type Bicuculline and Npn-2 −/− Bicuculline, P = 0.04; two-way ANOVA with Sidak’s multiple comparison test, two-way ANOVA interaction term: P = 0.0213). (C) Surface synaptic GluA1 immunostaining in wild type and Npn-2 −/− cortical neuron cultures (E18, 14 DIV) treated for 48 additional hours with bicuculline or control media. Synaptic GluA1 was identified by co-staining with a PSD95 antibody. (D) Quantification of synaptic GluA1 in wild type and Npn-2 −/− cortical cultures relative to control cultures following bicuculline treatments (n = 35–40 neurons for each condition; see STAR Methods). *P

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: Immunostaining, Staining

    Sema3F and Neuronal Activity Regulate the Interaction between Npn-2 and GluA1 (A) Regulation of the interaction between HA-GluA1 and FLAG-Npn-2 in HEK293T cells by Sema3F. HEK293T cells transfected with HA-GluA1, FLAG-Npn-2, and Myc-PlexA3 constructs were treated with AP-Sema3F (5 nM) for the indicated times. Npn-2 was co-immunoprecipitated with GluA1 from the transfected cell lysates using an HA antibody. (B) Quantification of Npn-2 coimmunoprecipitations with GluA1 from transfected HEK293T cell lysates following Sema3F treatment. (n = 3 experiments). For quantification, the intensity of coimmunoprecipitated Npn-2 was normalized to the intensity of Npn-2 input, and the value of the normalized control sample (0 min) was set as 100%. (C) Sema3F regulation of the interaction between GluA1 and Npn-2 in cortical neurons. 14 DIV cortical neurons were treated with 5 nM Sema3F for the indicated times, and cell lysates were collected and subjected to coimmunoprecipitation using a GluA1 antibody. (D) Quantification of the Npn-2 interaction with GluA1 upon Sema3F treatment relative to the untreated control, presented in C (n=3 experiments). Quantification was performed as described in B. (E) HEK293T cells were transfected with constructs expressing either wild type FLAG-Npn-2 or FLAG-Npn-2 Sema3F− together with HA-GluA1 and Myc-PlexA3. Transfected cells were treated with either AP or AP-Sema3F for 30 min. (F) Quantification of experiments in E, showing that Sema3F treatment fails to modulate the interaction between Npn-2 Sema3F− (Npn-2 lacking the ability to bind Sema3F) and GluA1 (n = 3 experiments). Quantification was performed as described in B. (G) Neuronal activity regulates the interaction between Npn-2 and GluA1. Cortical cultures (14 DIV) derived from wild type or Sema3F −/− embryos were treated with bicuculline or control media for 48 hrs; cell lysates were collected and subjected to co-immunoprecipitation with a GluA1 antibody. (H) Quantification of G. Coimmunoprecipitated Npn-2 following bicuculline treatment was quantified relative to the untreated sample. Quantification was performed as described in B (n = 3 experiments). *P

    Journal: Neuron

    Article Title: Neuropilin-2/PlexinA3 Receptors Associate with GluA1 and Mediate Sema3F-dependent Homeostatic Scaling in Cortical Neurons

    doi: 10.1016/j.neuron.2017.10.029

    Figure Lengend Snippet: Sema3F and Neuronal Activity Regulate the Interaction between Npn-2 and GluA1 (A) Regulation of the interaction between HA-GluA1 and FLAG-Npn-2 in HEK293T cells by Sema3F. HEK293T cells transfected with HA-GluA1, FLAG-Npn-2, and Myc-PlexA3 constructs were treated with AP-Sema3F (5 nM) for the indicated times. Npn-2 was co-immunoprecipitated with GluA1 from the transfected cell lysates using an HA antibody. (B) Quantification of Npn-2 coimmunoprecipitations with GluA1 from transfected HEK293T cell lysates following Sema3F treatment. (n = 3 experiments). For quantification, the intensity of coimmunoprecipitated Npn-2 was normalized to the intensity of Npn-2 input, and the value of the normalized control sample (0 min) was set as 100%. (C) Sema3F regulation of the interaction between GluA1 and Npn-2 in cortical neurons. 14 DIV cortical neurons were treated with 5 nM Sema3F for the indicated times, and cell lysates were collected and subjected to coimmunoprecipitation using a GluA1 antibody. (D) Quantification of the Npn-2 interaction with GluA1 upon Sema3F treatment relative to the untreated control, presented in C (n=3 experiments). Quantification was performed as described in B. (E) HEK293T cells were transfected with constructs expressing either wild type FLAG-Npn-2 or FLAG-Npn-2 Sema3F− together with HA-GluA1 and Myc-PlexA3. Transfected cells were treated with either AP or AP-Sema3F for 30 min. (F) Quantification of experiments in E, showing that Sema3F treatment fails to modulate the interaction between Npn-2 Sema3F− (Npn-2 lacking the ability to bind Sema3F) and GluA1 (n = 3 experiments). Quantification was performed as described in B. (G) Neuronal activity regulates the interaction between Npn-2 and GluA1. Cortical cultures (14 DIV) derived from wild type or Sema3F −/− embryos were treated with bicuculline or control media for 48 hrs; cell lysates were collected and subjected to co-immunoprecipitation with a GluA1 antibody. (H) Quantification of G. Coimmunoprecipitated Npn-2 following bicuculline treatment was quantified relative to the untreated sample. Quantification was performed as described in B (n = 3 experiments). *P

    Article Snippet: Primary antibodies: mouse-anti-GluA1 (4.9D- , mouse-anti-GluA2 -( )), mouse-anti-FLAG (M2, Sigma), mouse-anti-HA (12CA5, Roche), mouse-anti-HA (16B12, Biolegend), mouse-anti-Myc (Sigma), mouse-anti-PSD95 (NeuroMab), rabbit-anti-Myc (Cell Signaling), rabbit-anti-Npn-2 (Cell Signaling), rabbit-anti-HA (Sigma), rabbit-anti-HA (Cell Signaling, monoclonal), chicken-anti-GFP (Aves), guinea pig anti-GluA1 (Alomone Labs), guinea pig anti-vGlut1 (Millipore), rat-anti-Ctip2 (Abcam) and rat-anti-HA (3F10, Roche).

    Techniques: Activity Assay, Transfection, Construct, Immunoprecipitation, Expressing, Derivative Assay

    Immunostaining of AMPA receptor subunits in the olfactory bulb. ( a ) GluA2 immunoreactivity (green) was detected in the external plexiform layer (EPL) and in cell bodies surrounding glomeruli. Glomeruli are indicated by asterisks. Moderate GluA2 immunoreactivity was also found in astrocytes highlighted by GFAP immunoreactivity (red), as indicated by yellow pixels in the merged image. Arrows point to astrocyte structures that were colabeled with GluA immunoreactivity. Nuclei were stained with Hoechst 33342 (blue). ( b ) GluA1 and GFAP colocalization. ( c ) GluA4 and GFAP colocalization. Scale bars: 20 μm.

    Journal: Scientific Reports

    Article Title: Ca2+-permeable AMPA receptors in mouse olfactory bulb astrocytes

    doi: 10.1038/srep44817

    Figure Lengend Snippet: Immunostaining of AMPA receptor subunits in the olfactory bulb. ( a ) GluA2 immunoreactivity (green) was detected in the external plexiform layer (EPL) and in cell bodies surrounding glomeruli. Glomeruli are indicated by asterisks. Moderate GluA2 immunoreactivity was also found in astrocytes highlighted by GFAP immunoreactivity (red), as indicated by yellow pixels in the merged image. Arrows point to astrocyte structures that were colabeled with GluA immunoreactivity. Nuclei were stained with Hoechst 33342 (blue). ( b ) GluA1 and GFAP colocalization. ( c ) GluA4 and GFAP colocalization. Scale bars: 20 μm.

    Article Snippet: Slices were cut with a vibratome (VT1000, Leica) and incubated with the primary antibodies anti-GluA1 (guinea pig; 1:200; Alomone Labs), anti-GluA2 (rabbit; 1:200; Millipore, Darmstadt, Germany), anti-GluA4 (rabbit; 1:200; Millipore), anti-GFAP (rabbit, 1:000, Dako, Hamburg, Germany), anti-GFAP (chicken; 1:500; Abcam) and anti-GFP (chicken, 1:500, SySy, Göttingen, Germany).

    Techniques: Immunostaining, Staining