glua1  (Alomone Labs)


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    Structured Review

    Alomone Labs glua1
    CRIPT partially colocalizes <t>GluA1-positive</t> and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.
    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
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    Images

    1) Product Images from "SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo"

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    Journal: eNeuro

    doi: 10.1523/ENEURO.0175-17.2017

    CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.
    Figure Legend Snippet: CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.

    Techniques Used: Staining

    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p
    Figure Legend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Techniques Used: Infection, Western Blot

    2) Product Images from "A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance"

    Article Title: A CDC42EP4/septin-based perisynaptic glial scaffold facilitates glutamate clearance

    Journal: Nature Communications

    doi: 10.1038/ncomms10090

    Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).
    Figure Legend Snippet: Morphological analysis of the neuronal and glial components in Cdc42ep4 fl/fl and Cdc42ep4 −/− cerebellar cortices. ( a ) Double-label IF of WT and KO cerebellar cortices for a Purkinje cell marker Car8 (red) and a parallel fibre (that is, granule cell) marker VGluT1 (top, green) or a climbing fibre marker VGluT2 (bottom, green). No obvious morphological anomaly, including aberrant CF–PC innervation, was found in the major neuronal components of KO-derived samples. Scale bar, 20 μm. ( b ) Transmission electron microscopy images of WT and KO molecular layers. No obvious ultrastructural difference was found between the genotypes. PF, parallel fibre terminal or bouton. PC, dendritic spine of Purkinje cell. Bergmann glial processes are tinted. Scale bar, 200 nm. ( c ) Cumulative histogram of PSD length of the PF–PC synapses, showing no significant difference between the genotypes ( n =92 synapses from two littermates for each genotype, NS, P > 0.05 by Kolmogorov–Smirnov test). ( d ) Quantitative immunoblot of WT and KO cerebellar PSD fractions for GluA1, GluA2 and GluA4 (the major subunits of the AMPARs), each normalized with PSD-95. There was no significant quantitative difference by genotype ( n =3, NS, P > 0.05 by t -test).

    Techniques Used: Marker, Derivative Assay, Transmission Assay, Electron Microscopy

    3) Product Images from "Selective Regulation of GluA Subunit Synthesis and AMPA Receptor-Mediated Synaptic Function and Plasticity by the Translation Repressor 4E-BP2 in Hippocampal Pyramidal Cells"

    Article Title: Selective Regulation of GluA Subunit Synthesis and AMPA Receptor-Mediated Synaptic Function and Plasticity by the Translation Repressor 4E-BP2 in Hippocampal Pyramidal Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.3264-12.2013

    Selective increase in translation of GluA1 and GluA2 subunits in 4E-BP2 −/− mice. A , Polysome profile analysis of hippocampi from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mice. No change is observed in the general translation profile as measured by the 254 nm absorbance of different fractions. The 40, 60, 80 s, and polysome fractions are shown. B , Quantitative RT-PCR of polysome fractions from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) hippocampi. The relative abundance of GluA1, GluA2, GluN1, GluN2A/B, PSD95 (Dlg4), and Arc mRNAs is measured with quantitative RT-PCR across the different fractions of the polysome profiles from 4E-BP2 −/− and wild-type mouse hippocampi ( n = 4), showing selective shift of GluA1 and GluA2 mRNA to heavier polysome fractions. Polysome fractions are highlighted in pink. C , Representative Western blot analysis ( n = 3) of total (crude) and synaptosomal (Syn.) fraction extracts from 4E-BP2 −/− (−/−) and wild-type (+/+) mice. Lysates are probed for GluA1, GluA2, GluN1, GluN2A, GluN2B, PSD95, and Arc. GFAP and histone H3 are markers for the purity of the synaptosomes, whereas actin is used as loading control. Note selective increase in expression of GluA1 and GluA2 in 4E-BP2 −/− relative to wild-type mice. D , Total mRNA amounts of GluA1 ( Gria1 ), GluA2 ( Gria2 ), GluN1 ( Grin1 ), GluN2A ( Grin2a ), GluN2B ( Grin2b ), Arc ( Arg3.1 ), and PSD95 ( Dlg4 ) in hippocampal lysates from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mice. No change is observed in the total amounts ( n = 4). E , Quantification of Western blot analysis from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mouse hippocampi. Relative protein amounts of GluA1 (increase crude: 34.3 ± 9.2%; Syn.: 31.4 ± 7.1%), GluA2 (increase Syn.: 38.7 ± 5.3%), GluN1, GluN2A, GluN2B, Arc, and PSD95 normalized to the actin signal are shown. * p
    Figure Legend Snippet: Selective increase in translation of GluA1 and GluA2 subunits in 4E-BP2 −/− mice. A , Polysome profile analysis of hippocampi from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mice. No change is observed in the general translation profile as measured by the 254 nm absorbance of different fractions. The 40, 60, 80 s, and polysome fractions are shown. B , Quantitative RT-PCR of polysome fractions from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) hippocampi. The relative abundance of GluA1, GluA2, GluN1, GluN2A/B, PSD95 (Dlg4), and Arc mRNAs is measured with quantitative RT-PCR across the different fractions of the polysome profiles from 4E-BP2 −/− and wild-type mouse hippocampi ( n = 4), showing selective shift of GluA1 and GluA2 mRNA to heavier polysome fractions. Polysome fractions are highlighted in pink. C , Representative Western blot analysis ( n = 3) of total (crude) and synaptosomal (Syn.) fraction extracts from 4E-BP2 −/− (−/−) and wild-type (+/+) mice. Lysates are probed for GluA1, GluA2, GluN1, GluN2A, GluN2B, PSD95, and Arc. GFAP and histone H3 are markers for the purity of the synaptosomes, whereas actin is used as loading control. Note selective increase in expression of GluA1 and GluA2 in 4E-BP2 −/− relative to wild-type mice. D , Total mRNA amounts of GluA1 ( Gria1 ), GluA2 ( Gria2 ), GluN1 ( Grin1 ), GluN2A ( Grin2a ), GluN2B ( Grin2b ), Arc ( Arg3.1 ), and PSD95 ( Dlg4 ) in hippocampal lysates from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mice. No change is observed in the total amounts ( n = 4). E , Quantification of Western blot analysis from 4E-BP2 −/− and wild-type (4E-BP2 +/+ ) mouse hippocampi. Relative protein amounts of GluA1 (increase crude: 34.3 ± 9.2%; Syn.: 31.4 ± 7.1%), GluA2 (increase Syn.: 38.7 ± 5.3%), GluN1, GluN2A, GluN2B, Arc, and PSD95 normalized to the actin signal are shown. * p

    Techniques Used: Mouse Assay, Quantitative RT-PCR, Western Blot, Expressing

    4) Product Images from "Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission"

    Article Title: Cholesterol modulates presynaptic and postsynaptic properties of excitatory synaptic transmission

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69454-5

    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p
    Figure Legend Snippet: Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or GluN2B (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p

    Techniques Used: Marker

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    Alomone Labs polyclonal anti glua1 antibody
    ADPDZ3 expression reduces surface glutamate receptor 1 <t>(GluA1)</t> in dendrites. Cultured rat hippocampal neurons were infected with Sindbis viruses encoding GFP or GFP-ADPDZ3 and incubated for 9 h to allow expression. The cultures were then subjected to immunostaining using <t>polyclonal</t> GluA1 antibody and Cy3-conjugated anti-mouse IgG antibody. They were visualized using confocal microscopy. a , b Representative images of expressed neurons and selected dendrites in the analyses. Dotted boxes indicate analyzed dendrites. Scale bar: 20 μm. c , d ADPDZ3 expression reduced the number of surface GluA1 particles (GFP: 100.00% ± 7.83%, n = 18, 1486 μm; ADPDZ3: 66.50% ± 6.21%, n = 14, 1254 μm; ** P
    Polyclonal Anti Glua1 Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Alomone Labs glur1 subunit
    Impairment of DHPG-induced LTD and reduction in cell surface expression of the AMPAR subunit <t>GluR1</t> in shShank3-treated hippocampal neurons. A , examples of mEPSCs recorded immediately before ( left ) and 30 min after ( right ) DHPG application. Miniature EPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μ m TTX and 50 μ m picrotoxin. To isolate mGluR-mediated long term depression, 50 μ m APV was co-applied with 100 μ m DHPG or applied alone as a control. A , top , in shCtrl-treated cultures, 100 μ m DHPG elicited a persistent decrease in mEPSC frequency. A , bottom , in shShank3-treated neurons, 100 μ m DHPG did not induce a decrease in mEPSC frequency. B , histogram showing mean ± S.E. % mEPSC amplitude in shCtrl or in shShank3-treated neurons stimulated with DHPG. C , effect of DHPG on mEPSC frequency in shCtrl ( black circles , n = 7 neurons) and in shShank3-treated neurons ( gray circles , n = 7 neurons). Data presented are mean ± S.E. from three culture preparations. The frequency of mEPSCs during the first 5 min of recordings ( i.e. before application of DHPG) was set to 100%. D , summary plot showing changes in mean mEPSC frequency 30–35 min after application of DHPG; *, p
    Glur1 Subunit, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ADPDZ3 expression reduces surface glutamate receptor 1 (GluA1) in dendrites. Cultured rat hippocampal neurons were infected with Sindbis viruses encoding GFP or GFP-ADPDZ3 and incubated for 9 h to allow expression. The cultures were then subjected to immunostaining using polyclonal GluA1 antibody and Cy3-conjugated anti-mouse IgG antibody. They were visualized using confocal microscopy. a , b Representative images of expressed neurons and selected dendrites in the analyses. Dotted boxes indicate analyzed dendrites. Scale bar: 20 μm. c , d ADPDZ3 expression reduced the number of surface GluA1 particles (GFP: 100.00% ± 7.83%, n = 18, 1486 μm; ADPDZ3: 66.50% ± 6.21%, n = 14, 1254 μm; ** P

    Journal: Molecular Brain

    Article Title: Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

    doi: 10.1186/s13041-019-0520-x

    Figure Lengend Snippet: ADPDZ3 expression reduces surface glutamate receptor 1 (GluA1) in dendrites. Cultured rat hippocampal neurons were infected with Sindbis viruses encoding GFP or GFP-ADPDZ3 and incubated for 9 h to allow expression. The cultures were then subjected to immunostaining using polyclonal GluA1 antibody and Cy3-conjugated anti-mouse IgG antibody. They were visualized using confocal microscopy. a , b Representative images of expressed neurons and selected dendrites in the analyses. Dotted boxes indicate analyzed dendrites. Scale bar: 20 μm. c , d ADPDZ3 expression reduced the number of surface GluA1 particles (GFP: 100.00% ± 7.83%, n = 18, 1486 μm; ADPDZ3: 66.50% ± 6.21%, n = 14, 1254 μm; ** P

    Article Snippet: The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).

    Techniques: Expressing, Cell Culture, Infection, Incubation, Immunostaining, Confocal Microscopy

    Complexes of PSD-95-KIF5 colocalized with GluA1 particles in dendrites. Cultured hippocampal neurons were transfected with PSD-95-GFP constructs and incubated for days. The cultures were immunostained with monoclonal anti-PSD-95 antibody and polyclonal GluA1 antibody; they were subsequently stained with C3-conjugated anti-mouse IgG and Alexa−Fluor® 647 anti-rabbit IgG antibody. a Representative images of immunostaining in the first row. Each image was merged to show colocalization in the second row. A colocalized image of PSD-95 and KIF5 was collated with the image of GluA1. The colocalized points appeared white. Scale bar: 20 μm. b Boxed dendrites were enlarged to see the colocalization of GluA1 with the complex of PSD-95-KIF5A. c Staufen expression increased the association of PSD-95 and KIF5A. HA-PSD-95 and FLAG- KIF5A were cotransfected with 1, 2, or 3 μg of Myc-Staufen, or without Myc-Staufen as a control. After immunoprecipitation using anti-FLAG antibody or mouse IgG, immunoprecipitates were analyzed by Western blotting using anti-HA antibody. The lower panel shows expression of each group. d Quantified data of Western blot analyses (0 μg of Staufen: 100.0% ± 0.00%, n = 3; 1 μg of Staufen: 141.6% ± 16.96%, n = 3; 2 μg of Staufen: 192.3% ± 6.59%; 3 μg of Staufen; 274.4% ± 42.30%, n = 3). N values indicate the number of independent experiments. e Schematic diagram showing GluA1-containing vesicle transport mediated by PSD-95-KIF5A complex in dendrites. TARP: transmembrane AMPA receptor regulatory protein

    Journal: Molecular Brain

    Article Title: Postsynaptic density protein 95 (PSD-95) is transported by KIF5 to dendritic regions

    doi: 10.1186/s13041-019-0520-x

    Figure Lengend Snippet: Complexes of PSD-95-KIF5 colocalized with GluA1 particles in dendrites. Cultured hippocampal neurons were transfected with PSD-95-GFP constructs and incubated for days. The cultures were immunostained with monoclonal anti-PSD-95 antibody and polyclonal GluA1 antibody; they were subsequently stained with C3-conjugated anti-mouse IgG and Alexa−Fluor® 647 anti-rabbit IgG antibody. a Representative images of immunostaining in the first row. Each image was merged to show colocalization in the second row. A colocalized image of PSD-95 and KIF5 was collated with the image of GluA1. The colocalized points appeared white. Scale bar: 20 μm. b Boxed dendrites were enlarged to see the colocalization of GluA1 with the complex of PSD-95-KIF5A. c Staufen expression increased the association of PSD-95 and KIF5A. HA-PSD-95 and FLAG- KIF5A were cotransfected with 1, 2, or 3 μg of Myc-Staufen, or without Myc-Staufen as a control. After immunoprecipitation using anti-FLAG antibody or mouse IgG, immunoprecipitates were analyzed by Western blotting using anti-HA antibody. The lower panel shows expression of each group. d Quantified data of Western blot analyses (0 μg of Staufen: 100.0% ± 0.00%, n = 3; 1 μg of Staufen: 141.6% ± 16.96%, n = 3; 2 μg of Staufen: 192.3% ± 6.59%; 3 μg of Staufen; 274.4% ± 42.30%, n = 3). N values indicate the number of independent experiments. e Schematic diagram showing GluA1-containing vesicle transport mediated by PSD-95-KIF5A complex in dendrites. TARP: transmembrane AMPA receptor regulatory protein

    Article Snippet: The cultures were pretreated using the preblock solution (2% BSA, 0.08 TritonX-100 in PBS) for 1 h and each antibody was directly added to the preblock solution for 2 h. The following antibodies were used for staining, each at a dilution of 1:50; monoclonal anti-PSD-95 antibody (clone 6G6-1C9; Affinity Bioreagents, Golden, CO, USA), polyclonal anti-PSD-95 antibody (Cell Signaling, Danvers, MA, USA), monoclonal anti-kinesin antibody (Clone: H2; Millipore, Temecula, CA, USA), polyclonal anti-synapsin I antibody (Millipore), polyclonal anti-GluA1 antibody (Upstate, Lake Placid, NY), polyclonal anti-GluA1 antibody (Alomone Labs, Jerusalem, Israel) for surface GluA1.The following antibodies were used for secondary staining, each at a dilution of 1:200: Alexa Fluor® 488 anti-rabbit IgG antibody (Molecular Probes, Eugene, OR, USA), Cy3-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), Cy3-conjugated anti-rabbit IgG antibody (Jackson ImmunoResearch Laboratories), and Alexa Fluor® 647 anti-rabbit IgG antibody (Molecular Probes).

    Techniques: Cell Culture, Transfection, Construct, Incubation, Staining, Immunostaining, Expressing, Immunoprecipitation, Western Blot

    CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.

    Journal: eNeuro

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    doi: 10.1523/ENEURO.0175-17.2017

    Figure Lengend Snippet: CRIPT partially colocalizes GluA1-positive and SAP97-positive puncta on dendrites. Mixed spinal cord cultures at DIV21 were immunocytochemically analyzed. The upper set of panels display images of HA-CRIPT and EGFP-SAP97, and the lower set of panels display images of HA-CRIPT and GluA1. For the HA-CRIPT/EGFP-SAP97 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), EGFP-SAP97 (green), and merge. As in Figure 3 , HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. EGFP-SAP97 is similarly distributed but appears more punctate. In the merge image, extensive colocalization is seen (yellow) both in the soma and the dendritic tree. Scale bar: 30 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree, including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. EGFP-SAP97 is clearly more punctate than HA-CRIPT and in the merge image areas of colocalization are seen (yellow, denoted with > ). EGFP-SAP97 appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 10 μm. For the HA-CRIPT/GluA1 panels, the first row of panels shows immunocytochemical staining for HA-CRIPT (red), GluA1 (green), and merge. Again, HA-CRIPT immunoreactivity is seen within the soma and throughout the dendritic tree. GluA1 is seen exclusively as puncta. In the merge image, colocalization is seen (yellow) in the dendritic tree. Scale bar: 20 μm. Beneath each of the lower power images are higher magnification images of the area outlined in a white box. In the high-power HA-CRIPT panel, immunocytochemically positive material is again seen in various morphologies within the dendritic tree including small or large round puncta and elongated dendritic shaft entities. HA-CRIPT appears inhomogenously within dendritic outgrowths that may represent spines or filopodia. GluA1 is exclusively punctate and in the merge image areas of colocalization are seen (yellow, denoted with > ). GluA1, like EGFP-SAP97, appears enriched at along the edges of HA-CRIPT immunoreactivity. Scale bar: 4.0 μm.

    Article Snippet: The following antibodies were used for immunocytochemistry: extracellular epitopes of GluA1 (Alomone Labs, catalog #AGC004), HA-tag [BioLegend, 16B11, catalog #901512 (mouse)], HA-tag [Santa Cruz Biotechnology, Y-11 (rabbit), catalog #SC-805], and synaptophysin (Sigma-Aldrich, catalog #SAB4502906).

    Techniques: Staining

    CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Journal: eNeuro

    Article Title: SAP97 Binding Partner CRIPT Promotes Dendrite Growth In Vitro and In Vivo

    doi: 10.1523/ENEURO.0175-17.2017

    Figure Lengend Snippet: CRIPT knockdown leads to a selective reduction in the abundance of GluA1 and SAP97. Mixed spinal cord cultures were infected with HSV engineered to express a miRNA targeting CRIPT or a scrambled control. Two days later, lysates were prepared and subjected to Western blottings. No more than six independent experiments were performed for the quantitative image analysis. CRIPT knockdown leads to a reduction in GluA1 and SAP97 abundance and no effect on the abundance of GluA2, GluA4, NR1, NR2A, NR2B, or PSD95. Representative images of Western blottings with actin loading controls are shown and quantification of band intensity in the bar graphs below; *significant difference between groups, p

    Article Snippet: The following antibodies were used for immunocytochemistry: extracellular epitopes of GluA1 (Alomone Labs, catalog #AGC004), HA-tag [BioLegend, 16B11, catalog #901512 (mouse)], HA-tag [Santa Cruz Biotechnology, Y-11 (rabbit), catalog #SC-805], and synaptophysin (Sigma-Aldrich, catalog #SAB4502906).

    Techniques: Infection, Western Blot

    SHP2 D61G overexpression at DIV 12 increases surface AMPA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV 12. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ** P

    Journal: Neuroscience letters

    Article Title: Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

    doi: 10.1016/j.neulet.2017.03.036

    Figure Lengend Snippet: SHP2 D61G overexpression at DIV 12 increases surface AMPA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV 12. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ** P

    Article Snippet: The following antibodies were used: polyclonal anti-GluN1 (extracellular) antibody (#AGC-001, alomone labs, Jerusalem, Israel), polyclonal anti-GluA1 (extracellular) antibody (#AGC-004, alomone labs), Cy™3-conjugated goat anti-rabbit IgG antibody (#111–165-003, Jackson ImmunoResearch Lab, West Grove, PA).0Images were acquired by using confocal microscope (Zeiss 710, Carl Zeiss, Oberkochen, Germany) and analyzed by using NIH ImageJ software (ver.

    Techniques: Over Expression, Expressing, Immunostaining, Cell Culture, Staining

    SHP2 D61G overexpression at DIV 6 increases surface NMDA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV6 by using a bicistronic Sindbis virus vector. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ns, not significant. (D) Bar graph showing the average size (left) or the number (right) of GluA1-stained particles per 10 μm of dendrites. Unpaired t -test, * P

    Journal: Neuroscience letters

    Article Title: Noonan syndrome-associated SHP2 mutation differentially modulates the expression of postsynaptic receptors according to developmental maturation

    doi: 10.1016/j.neulet.2017.03.036

    Figure Lengend Snippet: SHP2 D61G overexpression at DIV 6 increases surface NMDA receptor expression. (A-B) Representative images of surface GluN1 (A) or GluA1 (B) receptor immunostaining. GFP alone (upper) or SHP2 D61G and GFP (lower) were expressed in cultured hippocampal neuron at DIV6 by using a bicistronic Sindbis virus vector. Scale bar, 20 μm. (C) Bar graph showing the average size (left) or the number (right) of GluN1-stained particles per 10 μm of dendrites. Unpaired t -test, ns, not significant. (D) Bar graph showing the average size (left) or the number (right) of GluA1-stained particles per 10 μm of dendrites. Unpaired t -test, * P

    Article Snippet: The following antibodies were used: polyclonal anti-GluN1 (extracellular) antibody (#AGC-001, alomone labs, Jerusalem, Israel), polyclonal anti-GluA1 (extracellular) antibody (#AGC-004, alomone labs), Cy™3-conjugated goat anti-rabbit IgG antibody (#111–165-003, Jackson ImmunoResearch Lab, West Grove, PA).0Images were acquired by using confocal microscope (Zeiss 710, Carl Zeiss, Oberkochen, Germany) and analyzed by using NIH ImageJ software (ver.

    Techniques: Over Expression, Expressing, Immunostaining, Cell Culture, Plasmid Preparation, Staining

    Impairment of DHPG-induced LTD and reduction in cell surface expression of the AMPAR subunit GluR1 in shShank3-treated hippocampal neurons. A , examples of mEPSCs recorded immediately before ( left ) and 30 min after ( right ) DHPG application. Miniature EPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μ m TTX and 50 μ m picrotoxin. To isolate mGluR-mediated long term depression, 50 μ m APV was co-applied with 100 μ m DHPG or applied alone as a control. A , top , in shCtrl-treated cultures, 100 μ m DHPG elicited a persistent decrease in mEPSC frequency. A , bottom , in shShank3-treated neurons, 100 μ m DHPG did not induce a decrease in mEPSC frequency. B , histogram showing mean ± S.E. % mEPSC amplitude in shCtrl or in shShank3-treated neurons stimulated with DHPG. C , effect of DHPG on mEPSC frequency in shCtrl ( black circles , n = 7 neurons) and in shShank3-treated neurons ( gray circles , n = 7 neurons). Data presented are mean ± S.E. from three culture preparations. The frequency of mEPSCs during the first 5 min of recordings ( i.e. before application of DHPG) was set to 100%. D , summary plot showing changes in mean mEPSC frequency 30–35 min after application of DHPG; *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Importance of Shank3 Protein in Regulating Metabotropic Glutamate Receptor 5 (mGluR5) Expression and Signaling at Synapses *

    doi: 10.1074/jbc.M111.258384

    Figure Lengend Snippet: Impairment of DHPG-induced LTD and reduction in cell surface expression of the AMPAR subunit GluR1 in shShank3-treated hippocampal neurons. A , examples of mEPSCs recorded immediately before ( left ) and 30 min after ( right ) DHPG application. Miniature EPSCs were recorded at a holding potential of −60 mV in the presence of 0.5 μ m TTX and 50 μ m picrotoxin. To isolate mGluR-mediated long term depression, 50 μ m APV was co-applied with 100 μ m DHPG or applied alone as a control. A , top , in shCtrl-treated cultures, 100 μ m DHPG elicited a persistent decrease in mEPSC frequency. A , bottom , in shShank3-treated neurons, 100 μ m DHPG did not induce a decrease in mEPSC frequency. B , histogram showing mean ± S.E. % mEPSC amplitude in shCtrl or in shShank3-treated neurons stimulated with DHPG. C , effect of DHPG on mEPSC frequency in shCtrl ( black circles , n = 7 neurons) and in shShank3-treated neurons ( gray circles , n = 7 neurons). Data presented are mean ± S.E. from three culture preparations. The frequency of mEPSCs during the first 5 min of recordings ( i.e. before application of DHPG) was set to 100%. D , summary plot showing changes in mean mEPSC frequency 30–35 min after application of DHPG; *, p

    Article Snippet: To estimate cell surface expression of the GluR1 subunit of AMPA receptors and its down-regulation by DHPG , untreated or DHPG-treated cultures were briefly washed with PBS and incubated with antibody against an extracellular epitope of the GluR1 subunit (Alomone Labs, agc-004, 16 μg/ml) for 15 min at 37 °C.

    Techniques: Expressing