guinea pig anti glur1 glua1 extracellular antibody  (Alomone Labs)


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    Alomone Labs guinea pig anti glur1 glua1 extracellular antibody
    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics <t>GluA1-containing</t> AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer
    Guinea Pig Anti Glur1 Glua1 Extracellular Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition"

    Article Title: Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

    Journal: Molecular Brain

    doi: 10.1186/s13041-021-00848-w

    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer
    Figure Legend Snippet: Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer

    Techniques Used: Expressing, Mutagenesis

    Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p
    Figure Legend Snippet: Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p

    Techniques Used: Inhibition, Cell Culture, In Silico, Staining

    2) Product Images from "Nogo-A Modulates the Synaptic Excitation of Hippocampal Neurons in a Ca2+-Dependent Manner"

    Article Title: Nogo-A Modulates the Synaptic Excitation of Hippocampal Neurons in a Ca2+-Dependent Manner

    Journal: Cells

    doi: 10.3390/cells10092299

    Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p
    Figure Legend Snippet: Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p

    Techniques Used: Immunolabeling, Immunofluorescence, Marker, Blocking Assay, Fluorescence

    3) Product Images from "Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition"

    Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

    Journal: bioRxiv

    doi: 10.1101/2021.01.18.427223

    Acute MLi-2 treatment reduces LRRK2 kinase activity in murine brain with no effect on protein expression A) Western blot of whole brain lysate following acute MLi-2 treatment were probed for LRRK2, LRRK2 phospho-S935, GluA1, VPS35, VGluTl, Rab10, Rab10 phospho-T73, and β-actin. B) There were no significant effects of genotype or treatment on LRRK2 levels (2-way ANOVA genotype x treatment p =0.93; genotype p =0.90; treatment p =0.24). C) ML12 treatment significantly reduced pLRRK2 in all genotypes (2-way ANOVA treatment p
    Figure Legend Snippet: Acute MLi-2 treatment reduces LRRK2 kinase activity in murine brain with no effect on protein expression A) Western blot of whole brain lysate following acute MLi-2 treatment were probed for LRRK2, LRRK2 phospho-S935, GluA1, VPS35, VGluTl, Rab10, Rab10 phospho-T73, and β-actin. B) There were no significant effects of genotype or treatment on LRRK2 levels (2-way ANOVA genotype x treatment p =0.93; genotype p =0.90; treatment p =0.24). C) ML12 treatment significantly reduced pLRRK2 in all genotypes (2-way ANOVA treatment p

    Techniques Used: Activity Assay, Expressing, Western Blot

    GluA1 protein expression levels are unaltered but dendritic cluster density is reduced in VKI A) Western blot of GluAi and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii). B) Co-immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta)(i). There was a significant reduction in GluA1 cluster density in homozygous VKI neurons (ii, ** p
    Figure Legend Snippet: GluA1 protein expression levels are unaltered but dendritic cluster density is reduced in VKI A) Western blot of GluAi and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii). B) Co-immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta)(i). There was a significant reduction in GluA1 cluster density in homozygous VKI neurons (ii, ** p

    Techniques Used: Expressing, Western Blot, Mouse Assay, Immunoprecipitation, Cell Culture

    Co-immunoprecipitation in cortical and striatal lysates from 3-month-old mice reveals novel neuronal VPS35 cargoes and no genotype effect on cargo binding. A) Western blot of cortical lysates and coIPs were probed for VPS35, D2R, GluA1, GluN1, GluA1, and GAPDH(i). There was no genotype effect on VPS35 levels or IP (ii-iii, 1-way ANOVA p > 0.99; Kruskal-Wallis p =0.62, respectively). NMDA-receptor subunit GluN1 association with retromer has not previously been published; the mutation did not affect GluN1 levels nor coIP with VPS35 (iv-v, Kruskal-Wallis p =0.76; p =0.44, respectively). D2-type dopamine receptors are a novel cargo; there was no significant genotype effect on D2R levels or CoIP with VPS35 (vi-vii, Kruskal-Wallis p =0.80; p =0.44, respectively). B) CoIP of cortical lysates in A probed for LRRK2 (i). There were no significant genotype effects on LRRK2 levels or association of LRRK2 with VPS35 by coIP (ii-iii Kruskal-Wallis p =0.76; p =0.52, respectively). C) Striatal lysates quantified as in A B (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii-iii, Kruskal-Wallis p =0.97; p =0.13, respectively); GluN1 levels or coIP (iv-v, Kruskal-Wallis p =0.51; p =0.42, respectively); D2R levels or coIP (vi-vii Kruskal-Wallis p =0.70; p =0.45, respectively); GluA1 levels or coIP (viii-ix, Kruskal-Wallis p =0.83; p =0.44, respectively); or LRRK2 levels or coIP (x-xi, Kruskal-Wallis p > 0.99; p =0.40, respectively). For all panels, n= number of experimental animals.
    Figure Legend Snippet: Co-immunoprecipitation in cortical and striatal lysates from 3-month-old mice reveals novel neuronal VPS35 cargoes and no genotype effect on cargo binding. A) Western blot of cortical lysates and coIPs were probed for VPS35, D2R, GluA1, GluN1, GluA1, and GAPDH(i). There was no genotype effect on VPS35 levels or IP (ii-iii, 1-way ANOVA p > 0.99; Kruskal-Wallis p =0.62, respectively). NMDA-receptor subunit GluN1 association with retromer has not previously been published; the mutation did not affect GluN1 levels nor coIP with VPS35 (iv-v, Kruskal-Wallis p =0.76; p =0.44, respectively). D2-type dopamine receptors are a novel cargo; there was no significant genotype effect on D2R levels or CoIP with VPS35 (vi-vii, Kruskal-Wallis p =0.80; p =0.44, respectively). B) CoIP of cortical lysates in A probed for LRRK2 (i). There were no significant genotype effects on LRRK2 levels or association of LRRK2 with VPS35 by coIP (ii-iii Kruskal-Wallis p =0.76; p =0.52, respectively). C) Striatal lysates quantified as in A B (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii-iii, Kruskal-Wallis p =0.97; p =0.13, respectively); GluN1 levels or coIP (iv-v, Kruskal-Wallis p =0.51; p =0.42, respectively); D2R levels or coIP (vi-vii Kruskal-Wallis p =0.70; p =0.45, respectively); GluA1 levels or coIP (viii-ix, Kruskal-Wallis p =0.83; p =0.44, respectively); or LRRK2 levels or coIP (x-xi, Kruskal-Wallis p > 0.99; p =0.40, respectively). For all panels, n= number of experimental animals.

    Techniques Used: Immunoprecipitation, Mouse Assay, Binding Assay, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay

    Rab10 does not colocalize with VPS35 or GluA1 in cortical neurites. A) There was no genotype effect on Rab10 cluster intensity (i); however, Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, p
    Figure Legend Snippet: Rab10 does not colocalize with VPS35 or GluA1 in cortical neurites. A) There was no genotype effect on Rab10 cluster intensity (i); however, Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, p

    Techniques Used: Mutagenesis

    4) 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

    5) 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

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    Alomone Labs guinea pig anti glur1 glua1 extracellular antibody
    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics <t>GluA1-containing</t> AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer
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    Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer

    Journal: Molecular Brain

    Article Title: Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

    doi: 10.1186/s13041-021-00848-w

    Figure Lengend Snippet: Working model of increased surface AMPAR expression in D620N heterozygous mutants. In wild-type cells, postsynaptic VPS35 traffics GluA1-containing AMPARs. At the drosophila neuromuscular junction, VPS35 localizes to large endocytic structures resembling bulk-endocytosed membrane, raising the possibility that it participates in SV regeneration [ 25 ]; however, in mammalian neurons it localizes to only a subset of terminals [ 22 ] and retromer deficiency has no effect on SV endo- or exocytosis [ 22 ] or neurotransmitter release [ 16 , 21 ]. Thus, we propose that presynaptic VPS35 may participate in recycling or retrograde transport of presynaptic receptors, channels, and/or SV proteins. In heterozygous neurons, there is increased abundance of endosomal structures positive for VPS35 and FAM21, accumulation of Rab11 + ve recycling endosomes, and increased surface expression of GluA1, thus we propose that the D620N mutation causes increased surface recycling of GluA1. Given the proposed presynaptic functions of retromer, we hypothesize that the observed increase in the probability of glutamate release is the result of either increased SV regeneration by retromer, or complex alterations to the recycling and axonal trafficking of presynaptic proteins by retromer

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Expressing, Mutagenesis

    Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p

    Journal: Molecular Brain

    Article Title: Endosomal traffic and glutamate synapse activity are increased in VPS35 D620N mutant knock-in mouse neurons, and resistant to LRRK2 kinase inhibition

    doi: 10.1186/s13041-021-00848-w

    Figure Lengend Snippet: Surface GluA1 is increased in VKI and altered by acute LRRK2 kinase inhibition. A GFP-filled (cyan) cultured cortical cells immunostained for MAP2 (blue; to ensure no permeabilization) and surface GluA1 (magenta) (i, left panel); in silico neurite outlines with only GluA1 staining displayed (i, right panel). There was a significant genotype effect on GluA1 cluster density, due to opposing effects on heterozygous and homozygous cells (ii, Kruskal–Wallis p

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Inhibition, Cell Culture, In Silico, Staining

    Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p

    Journal: Cells

    Article Title: Nogo-A Modulates the Synaptic Excitation of Hippocampal Neurons in a Ca2+-Dependent Manner

    doi: 10.3390/cells10092299

    Figure Lengend Snippet: Nogo-A regulates the synaptic insertion of calcium permeable-AMPARs. ( A , B ) Live-cell immunolabeling of surface AMPAR subunit (magenta) GluA1 ( A ) or GluA2 ( B ) followed by immunofluorescence for presynaptic marker synapsin (Syn1/2; green) and their merged images (bottom) in primary hippocampal neurons treated for 10 min either with the control (left) or the Nogo-A function-blocking (right) antibody. For illustration, all images underwent deconvolution and were equally increased in brightness and contrast by the same absolute values for visibility. Scale bar 2 μm. ( C , D ) Normalized data for density ( C ) and fluorescence intensity ( D ) of GluA1 immuno-positive puncta in hippocampal neurons treated with either control antibody (black, n = 40) or Nogo-A function-blocking antibody (red, n = 39) for 10 min. ( E ) Normalized values for the density of GluA1 clusters colocalized with Syn 1/2 immuno-positive puncta. ( F , G ) Normalized GluA2 cluster density ( F ) and fluorescence intensity ( G ) in hippocampal neurons upon 10 min application with control antibody (black, n = 36) or Nogo-A function-blocking antibody (red, n = 35). ( H ) Normalized density of GluA2 immuno-positive puncta colocalized with Syn 1/2. Data are presented as mean ± SEM. * p

    Article Snippet: In the case of the AMPA receptors, the anti-AMPAR 1 GluA1 (Alomone Labs, Jerusalem, Israel, Cat# AGP-009, 1:50) and anti-AMPAR 2 GluA2 (Alomone Labs, Cat# AGC-005, 1:50) were co-applied with the Nogo-A or control antibodies for 10 min. After completion of the treatment, the coverslips were rinsed with pre-warmed NB- medium and fixed with 4% paraformaldehyde (PFA) in phosphate buffer (PB containing in mM 50 NaH2 PO4 *2H2 O, 85 Na2 HPO4 *2H2 O) for 10 min at room temperature (RT).

    Techniques: Immunolabeling, Immunofluorescence, Marker, Blocking Assay, Fluorescence

    Acute MLi-2 treatment reduces LRRK2 kinase activity in murine brain with no effect on protein expression A) Western blot of whole brain lysate following acute MLi-2 treatment were probed for LRRK2, LRRK2 phospho-S935, GluA1, VPS35, VGluTl, Rab10, Rab10 phospho-T73, and β-actin. B) There were no significant effects of genotype or treatment on LRRK2 levels (2-way ANOVA genotype x treatment p =0.93; genotype p =0.90; treatment p =0.24). C) ML12 treatment significantly reduced pLRRK2 in all genotypes (2-way ANOVA treatment p

    Journal: bioRxiv

    Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

    doi: 10.1101/2021.01.18.427223

    Figure Lengend Snippet: Acute MLi-2 treatment reduces LRRK2 kinase activity in murine brain with no effect on protein expression A) Western blot of whole brain lysate following acute MLi-2 treatment were probed for LRRK2, LRRK2 phospho-S935, GluA1, VPS35, VGluTl, Rab10, Rab10 phospho-T73, and β-actin. B) There were no significant effects of genotype or treatment on LRRK2 levels (2-way ANOVA genotype x treatment p =0.93; genotype p =0.90; treatment p =0.24). C) ML12 treatment significantly reduced pLRRK2 in all genotypes (2-way ANOVA treatment p

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Activity Assay, Expressing, Western Blot

    GluA1 protein expression levels are unaltered but dendritic cluster density is reduced in VKI A) Western blot of GluAi and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii). B) Co-immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta)(i). There was a significant reduction in GluA1 cluster density in homozygous VKI neurons (ii, ** p

    Journal: bioRxiv

    Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

    doi: 10.1101/2021.01.18.427223

    Figure Lengend Snippet: GluA1 protein expression levels are unaltered but dendritic cluster density is reduced in VKI A) Western blot of GluAi and β-actin in cortical lysates of VKI mice (i) revealed no genotype effect on GluA1 protein levels (ii). B) Co-immunoprecipitation of GluA1 with VPS35 (i) revealed no genotype effect (ii). C) Cultured cortical neurons immunostained for MAP2 (blue), VPS35 (cyan), and GluA1 (magenta)(i). There was a significant reduction in GluA1 cluster density in homozygous VKI neurons (ii, ** p

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Expressing, Western Blot, Mouse Assay, Immunoprecipitation, Cell Culture

    Co-immunoprecipitation in cortical and striatal lysates from 3-month-old mice reveals novel neuronal VPS35 cargoes and no genotype effect on cargo binding. A) Western blot of cortical lysates and coIPs were probed for VPS35, D2R, GluA1, GluN1, GluA1, and GAPDH(i). There was no genotype effect on VPS35 levels or IP (ii-iii, 1-way ANOVA p > 0.99; Kruskal-Wallis p =0.62, respectively). NMDA-receptor subunit GluN1 association with retromer has not previously been published; the mutation did not affect GluN1 levels nor coIP with VPS35 (iv-v, Kruskal-Wallis p =0.76; p =0.44, respectively). D2-type dopamine receptors are a novel cargo; there was no significant genotype effect on D2R levels or CoIP with VPS35 (vi-vii, Kruskal-Wallis p =0.80; p =0.44, respectively). B) CoIP of cortical lysates in A probed for LRRK2 (i). There were no significant genotype effects on LRRK2 levels or association of LRRK2 with VPS35 by coIP (ii-iii Kruskal-Wallis p =0.76; p =0.52, respectively). C) Striatal lysates quantified as in A B (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii-iii, Kruskal-Wallis p =0.97; p =0.13, respectively); GluN1 levels or coIP (iv-v, Kruskal-Wallis p =0.51; p =0.42, respectively); D2R levels or coIP (vi-vii Kruskal-Wallis p =0.70; p =0.45, respectively); GluA1 levels or coIP (viii-ix, Kruskal-Wallis p =0.83; p =0.44, respectively); or LRRK2 levels or coIP (x-xi, Kruskal-Wallis p > 0.99; p =0.40, respectively). For all panels, n= number of experimental animals.

    Journal: bioRxiv

    Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

    doi: 10.1101/2021.01.18.427223

    Figure Lengend Snippet: Co-immunoprecipitation in cortical and striatal lysates from 3-month-old mice reveals novel neuronal VPS35 cargoes and no genotype effect on cargo binding. A) Western blot of cortical lysates and coIPs were probed for VPS35, D2R, GluA1, GluN1, GluA1, and GAPDH(i). There was no genotype effect on VPS35 levels or IP (ii-iii, 1-way ANOVA p > 0.99; Kruskal-Wallis p =0.62, respectively). NMDA-receptor subunit GluN1 association with retromer has not previously been published; the mutation did not affect GluN1 levels nor coIP with VPS35 (iv-v, Kruskal-Wallis p =0.76; p =0.44, respectively). D2-type dopamine receptors are a novel cargo; there was no significant genotype effect on D2R levels or CoIP with VPS35 (vi-vii, Kruskal-Wallis p =0.80; p =0.44, respectively). B) CoIP of cortical lysates in A probed for LRRK2 (i). There were no significant genotype effects on LRRK2 levels or association of LRRK2 with VPS35 by coIP (ii-iii Kruskal-Wallis p =0.76; p =0.52, respectively). C) Striatal lysates quantified as in A B (i). There were no genotype effects on VPS35 levels or pull by the antibody (ii-iii, Kruskal-Wallis p =0.97; p =0.13, respectively); GluN1 levels or coIP (iv-v, Kruskal-Wallis p =0.51; p =0.42, respectively); D2R levels or coIP (vi-vii Kruskal-Wallis p =0.70; p =0.45, respectively); GluA1 levels or coIP (viii-ix, Kruskal-Wallis p =0.83; p =0.44, respectively); or LRRK2 levels or coIP (x-xi, Kruskal-Wallis p > 0.99; p =0.40, respectively). For all panels, n= number of experimental animals.

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Immunoprecipitation, Mouse Assay, Binding Assay, Western Blot, Mutagenesis, Co-Immunoprecipitation Assay

    Rab10 does not colocalize with VPS35 or GluA1 in cortical neurites. A) There was no genotype effect on Rab10 cluster intensity (i); however, Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, p

    Journal: bioRxiv

    Article Title: Surface GluA1 and glutamatergic transmission are increased in cortical neurons of a VPS35 D620N knock-in mouse model of parkinsonism and altered by LRRK2 kinase inhibition

    doi: 10.1101/2021.01.18.427223

    Figure Lengend Snippet: Rab10 does not colocalize with VPS35 or GluA1 in cortical neurites. A) There was no genotype effect on Rab10 cluster intensity (i); however, Rab10 cluster density was increased in both mutant genotypes, falling just shy of statistical significance (ii, p

    Article Snippet: We used the following primary antibodies: GFP (Abcam ab1218); VPS35 (Abnova H00055737); VPS26 (a kind gift from J. Bonifacino, NICHD); FAM21C (Millipore ABT79); NEEP21/NSG1 (Genscript A01442); Rab11 (Abcam ab95375); MAP2 (Abcam ab5392); GluA1 (Millipore 05-855R); PSD95 (Thermo Scientific MA1-045); VGluT1 (Millipore AB5905); GluA1 extracellular (Millipore ABN241); Rab10 (Abcam 237703); and GluA1 (Alomone AGP-009).

    Techniques: Mutagenesis