rabbit anti nmdar 2b glun2b  (Alomone Labs)


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    Alomone Labs rabbit anti nmdar 2b glun2b
    Rabbit Anti Nmdar 2b Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nmdar 2b glun2b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nmdar 2b glun2b - by Bioz Stars, 2022-09
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    Alomone Labs anti nr2b
    Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total <t>NR2B</t> intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P
    Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nr2b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nr2b - by Bioz Stars, 2022-09
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    93
    Alomone Labs rabbit rb antibodies against glun2b
    ECM digestion increases <t>p1472-GluN2B</t> level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P
    Rabbit Rb Antibodies Against Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit rb antibodies against glun2b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit rb antibodies against glun2b - by Bioz Stars, 2022-09
    93/100 stars
      Buy from Supplier

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    Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Western Blot, Mouse Assay, Marker, Affinity Purification, Magnetic Beads

    Leptin-regulated NR2B Y1472 phosphorylation and surface expression is Fyn dependent. ( A ) Representative Western blot of HEK293T cells transfected with NR2B-V5, NR1, LepRb-myc, and either V5-Fyn or V5-DN Fyn and treated with leptin (50 nM, 2 hours). ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B-V5 intensity (n = 3). ( C ) Hippocampal neurons were transfected with Clover and EGFP-NR2B-V5 and either V5-Fyn or V5-DN Fyn ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP–NR2B. Quantification of immunostained EGFP-integrated signal density (n = 15). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: Leptin-regulated NR2B Y1472 phosphorylation and surface expression is Fyn dependent. ( A ) Representative Western blot of HEK293T cells transfected with NR2B-V5, NR1, LepRb-myc, and either V5-Fyn or V5-DN Fyn and treated with leptin (50 nM, 2 hours). ( B ) Quantification of pNR2B Y1472 intensity normalized to total NR2B-V5 intensity (n = 3). ( C ) Hippocampal neurons were transfected with Clover and EGFP-NR2B-V5 and either V5-Fyn or V5-DN Fyn ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP–NR2B. Quantification of immunostained EGFP-integrated signal density (n = 15). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Western Blot, Transfection

    LepRb directly interacts with NR2B. ( A ) Schematic of LepRb–BioID experiment with representative Western blot of NR2B-V5 immunoprecipitated from HEK293T cells expressing the designated BioID constructs and NR2B-V5 and NR1-Clover to the right. ( B ) Quantification of IP biotinylated NR2B-V5 intensity normalized to total NR2B-V5 intensity in the same lane (n = 3). ( C ) Schematic of NR2B–BioID experiment with representative Western blot of LepRb-V5 immunoprecipitated from HEK293T cells expressing designated BioID constructs and LepRb-V5 and NR1-Clover. ( D ) Representative Western blot of LepRb-myc immunoprecipitated from HEK293T cells stimulated with leptin (50 nM, 2 hours) and expressing LepRb-myc, NR2B-V5, and NR1-Clover. ( E ) Quantification of coimmunoprecipitated NR2B-V5 intensity normalized to immunoprecipitated LepRb-myc intensity from the same lane (n = 3). ( F ) Representative fluorescent images of hippocampal cultures expressing Flag-LepRb and Clover. Surface Flag-LepRb and endogenous surface NR2B were live immunostained after stimulation with leptin (50 nM, 2 hours). ( G ) Quantification of NR2B/Flag-LepRb puncta colocalization compared to total NR2B puncta. Colocalization experiments were repeated in 2 independent hippocampal culture preparations. All BioID experiments were stimulated with biotin (50 µM) at the time of transfection. All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: LepRb directly interacts with NR2B. ( A ) Schematic of LepRb–BioID experiment with representative Western blot of NR2B-V5 immunoprecipitated from HEK293T cells expressing the designated BioID constructs and NR2B-V5 and NR1-Clover to the right. ( B ) Quantification of IP biotinylated NR2B-V5 intensity normalized to total NR2B-V5 intensity in the same lane (n = 3). ( C ) Schematic of NR2B–BioID experiment with representative Western blot of LepRb-V5 immunoprecipitated from HEK293T cells expressing designated BioID constructs and LepRb-V5 and NR1-Clover. ( D ) Representative Western blot of LepRb-myc immunoprecipitated from HEK293T cells stimulated with leptin (50 nM, 2 hours) and expressing LepRb-myc, NR2B-V5, and NR1-Clover. ( E ) Quantification of coimmunoprecipitated NR2B-V5 intensity normalized to immunoprecipitated LepRb-myc intensity from the same lane (n = 3). ( F ) Representative fluorescent images of hippocampal cultures expressing Flag-LepRb and Clover. Surface Flag-LepRb and endogenous surface NR2B were live immunostained after stimulation with leptin (50 nM, 2 hours). ( G ) Quantification of NR2B/Flag-LepRb puncta colocalization compared to total NR2B puncta. Colocalization experiments were repeated in 2 independent hippocampal culture preparations. All BioID experiments were stimulated with biotin (50 µM) at the time of transfection. All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Construct, Transfection

    pNR2B Y1472 is necessary for leptin-stimulated spine formation. ( A ) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2B Y1472F -V5 ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP-NR2B. White bar = 20 µm. ( B ) Quantification of immunostained EGFP-integrated signal density (n = 23). ( C-E ) Hippocampal neurons that were transfected with a fluorescent Clover-βactin and EGFP-NR2B Y1472F -V5. Neurons were stimulated with leptin (50 nM) on DIV8, and on DIV11 to 12 spine density was measured by hand using ImageJ with the NeuronJ plugin ( C,D ), or electrophysiological recordings were performed ( E ). White bar = 5 µm. ( D ) Quantification of dendritic spine density from a minimum of 2 to 3 dendritic segments from 15 neurons. ( E ) Quantification of mEPSC frequency, amplitude, and decay time normalized to control condition (control: n = 32; control + leptin: n = 34; NR2B Y1472F : n = 33; NR2B Y1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Journal: Endocrinology

    Article Title: Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B

    doi: 10.1210/endocr/bqz030

    Figure Lengend Snippet: pNR2B Y1472 is necessary for leptin-stimulated spine formation. ( A ) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2B Y1472F -V5 ± leptin stimulation (50 nM, 2 hours) and live immunostained for surface EGFP-NR2B. White bar = 20 µm. ( B ) Quantification of immunostained EGFP-integrated signal density (n = 23). ( C-E ) Hippocampal neurons that were transfected with a fluorescent Clover-βactin and EGFP-NR2B Y1472F -V5. Neurons were stimulated with leptin (50 nM) on DIV8, and on DIV11 to 12 spine density was measured by hand using ImageJ with the NeuronJ plugin ( C,D ), or electrophysiological recordings were performed ( E ). White bar = 5 µm. ( D ) Quantification of dendritic spine density from a minimum of 2 to 3 dendritic segments from 15 neurons. ( E ) Quantification of mEPSC frequency, amplitude, and decay time normalized to control condition (control: n = 32; control + leptin: n = 34; NR2B Y1472F : n = 33; NR2B Y1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P

    Article Snippet: Neurons fixed and incubated with an anti-NR2B (1:100, Alomone Labs) ( ) and anti-Flag (1:250, Sigma Aldrich) ( ) antibody for 1 hour and then incubated with the appropriate Alexa Fluor secondary IgG antibody ( ) for 1 hour at room temperature.

    Techniques: Expressing, Transfection

    ECM digestion increases p1472-GluN2B level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P

    Journal: Scientific Reports

    Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

    doi: 10.1038/s41598-017-07003-3

    Figure Lengend Snippet: ECM digestion increases p1472-GluN2B level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P

    Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

    Techniques: Staining, CTL Assay, Western Blot, Binding Assay

    ECM removal enhances GluN2B-NMDAR mediated synaptic currents. ( A ) Example traces of NMDAR - mediated sEPCSs before and after Hya treatment in dissociated hippocampal cultures DIV21-24. ( B ) Amplitudes of single peaks show no significant differences between Hya treated or Hya plus Ifenprodil treated cultures (Ctl, −905.5 ± 179.4, n = 10; Hya, −776.2 ± 174.8, n = 10; Hya + Ifen, −758.2 ± 161.7, n = 11; average ± SEM; One-way ANOVA, P = 0.7991). ( C ) Average of single peaks before and after Hya treatment and after Ifenprodil application. Normalization of the amplitude illustrates the increased decay-time after Hya treatment (red line) in comparison to Ctl (black line). This can be restored after Ifenprodil application (green line). Ctl traces are identical. ( D ) Quantification of the area under the curve (AUC) of averaged and normalized events (left), which represent the total charge transfer revealed bigger charge transfer after ECM removal, which was reduced to control levels after blocking GluN2B-NMDAR with Ifen (Ctl, 1 ± 0.02, n = 10; Hya, 1.38 ± 0.09, n = 10; Hya + Ifenprodil, 0.98 ± 0.05, n = 11; average ± SEM; One-way ANOVA, P

    Journal: Scientific Reports

    Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

    doi: 10.1038/s41598-017-07003-3

    Figure Lengend Snippet: ECM removal enhances GluN2B-NMDAR mediated synaptic currents. ( A ) Example traces of NMDAR - mediated sEPCSs before and after Hya treatment in dissociated hippocampal cultures DIV21-24. ( B ) Amplitudes of single peaks show no significant differences between Hya treated or Hya plus Ifenprodil treated cultures (Ctl, −905.5 ± 179.4, n = 10; Hya, −776.2 ± 174.8, n = 10; Hya + Ifen, −758.2 ± 161.7, n = 11; average ± SEM; One-way ANOVA, P = 0.7991). ( C ) Average of single peaks before and after Hya treatment and after Ifenprodil application. Normalization of the amplitude illustrates the increased decay-time after Hya treatment (red line) in comparison to Ctl (black line). This can be restored after Ifenprodil application (green line). Ctl traces are identical. ( D ) Quantification of the area under the curve (AUC) of averaged and normalized events (left), which represent the total charge transfer revealed bigger charge transfer after ECM removal, which was reduced to control levels after blocking GluN2B-NMDAR with Ifen (Ctl, 1 ± 0.02, n = 10; Hya, 1.38 ± 0.09, n = 10; Hya + Ifenprodil, 0.98 ± 0.05, n = 11; average ± SEM; One-way ANOVA, P

    Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

    Techniques: CTL Assay, Blocking Assay

    ECM removal leads to increased surface expression of GluN2B in a β1 - integrin dependent manner. ( A ) Dissociated hippocampal cultures were treated with Hya over night and stained against the total amount of GluN2B and the dendritic marker Map2 (scale bar: 10 μm. ( B ) Total GluN2B expression is not affected by ECM removal (Dendrites: Ctl 1 ± 0.10, n = 30; Hya 0.89 ± 0.03, n = 30, P = 0.31; Synapses: Ctl: 1 ± 0.03, n = 30; Hya: 1.05 ± 0.03, n = 30, P = 0.27; average ± SEM; unpaired t-test). ( C ) Quantitative WB of lysed cortical cultures (DIV21) pretreated with Hya over night show no significant change in GluN2B immunoreactivity. ( D ) Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and stained against surface GluN2B (green) and the synaptic marker PSD-95 (scale bar: 10 μm). ( E ) Synaptic GluN2B surface expression at various time points after Hya treatment (Ctl: 1 ± 0.04, n = 24; Hya 1,5 h: 1.08 ± 0.04, n = 22, P = 0.76; Hya 3 h: 1.40 ± 0.09, n = 30, P = 0.0001; Hya 6 h: 1.41 ± 0.13, n = 9, P = 0.002; Hya 12 h: 1.35 ± 0.08, n = 8, P = 0.01; Hya 48 h: 1.18 ± 0.05, n = 8, P = 0.04 average ± SEM; One way-ANOVA, Dunnett’s Multiple Comparison Test). ( F,G ) GluN2B surface expression at synapses and dendrites increases after ECM degradation and can be restored by simultaneous application of the β1-integrin function blocking antibody CD29. ( F ) Synapses: Ctl: 1.0 ± 0.05, n = 68; Hya: 1.3 ± 0.05, n = 70; Hya + CD29: 0.93 ± 0.03, n = 51. ( G ) Dendrites: Ctl 1.00 ± 0.04, n = 36; Hya 1.78 ± 0.11, n = 35; Hya + CD29 0.96 ± 0.03, n = 34; average ± SEM; One-way ANOVA, P

    Journal: Scientific Reports

    Article Title: Hyaluronic acid based extracellular matrix regulates surface expression of GluN2B containing NMDA receptors

    doi: 10.1038/s41598-017-07003-3

    Figure Lengend Snippet: ECM removal leads to increased surface expression of GluN2B in a β1 - integrin dependent manner. ( A ) Dissociated hippocampal cultures were treated with Hya over night and stained against the total amount of GluN2B and the dendritic marker Map2 (scale bar: 10 μm. ( B ) Total GluN2B expression is not affected by ECM removal (Dendrites: Ctl 1 ± 0.10, n = 30; Hya 0.89 ± 0.03, n = 30, P = 0.31; Synapses: Ctl: 1 ± 0.03, n = 30; Hya: 1.05 ± 0.03, n = 30, P = 0.27; average ± SEM; unpaired t-test). ( C ) Quantitative WB of lysed cortical cultures (DIV21) pretreated with Hya over night show no significant change in GluN2B immunoreactivity. ( D ) Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and stained against surface GluN2B (green) and the synaptic marker PSD-95 (scale bar: 10 μm). ( E ) Synaptic GluN2B surface expression at various time points after Hya treatment (Ctl: 1 ± 0.04, n = 24; Hya 1,5 h: 1.08 ± 0.04, n = 22, P = 0.76; Hya 3 h: 1.40 ± 0.09, n = 30, P = 0.0001; Hya 6 h: 1.41 ± 0.13, n = 9, P = 0.002; Hya 12 h: 1.35 ± 0.08, n = 8, P = 0.01; Hya 48 h: 1.18 ± 0.05, n = 8, P = 0.04 average ± SEM; One way-ANOVA, Dunnett’s Multiple Comparison Test). ( F,G ) GluN2B surface expression at synapses and dendrites increases after ECM degradation and can be restored by simultaneous application of the β1-integrin function blocking antibody CD29. ( F ) Synapses: Ctl: 1.0 ± 0.05, n = 68; Hya: 1.3 ± 0.05, n = 70; Hya + CD29: 0.93 ± 0.03, n = 51. ( G ) Dendrites: Ctl 1.00 ± 0.04, n = 36; Hya 1.78 ± 0.11, n = 35; Hya + CD29 0.96 ± 0.03, n = 34; average ± SEM; One-way ANOVA, P

    Article Snippet: Antibodies and drugs The following commercial antibodies were used for Immunocytochemistry (ICC) and Western blot (WB) in the concentrations indicated: rabbit (rb) antibodies against GluN2B (alomone labs; ICC live staining: 1:200, fixed staining.

    Techniques: Expressing, Staining, Marker, CTL Assay, Western Blot, Blocking Assay

    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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-69454-5

    Figure Lengend 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

    Article Snippet: Immunocytochemistry Neurons were stained in non-permeabilized conditions with primary antibodies against extracellular epitopes of GluN2A, GluN2B or GluA1 (ACG-002 1:500, Alomone Labs , , ACG-003 1:500, Alomone Labs , , and PC246 1:1000, MERCK ) and then they were depleted of cholesterol.

    Techniques: Marker

    Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-69454-5

    Figure Lengend Snippet: Cholesterol depletion reduces the fraction of synaptic immobile NMDARs. ( A ) Surface NMDARs were detected using a QD-antibody complex directed against extracellular epitopes in GluN2A or GluN2B. Left, representative summed trajectories of NMDAR-QDs (red) acquired over a period of 25 s (20 Hz frame rate) in hippocampal neurons. Scale bar 5 µm. Right, representative examples of NMDAR reconstructed trajectories. ( B , C ) Diffusion coefficients for synaptic GluN2A-containing NMDARs and GluN2B-containing NMDARs in control and after cholesterol depletion (10 mM MβCD pretreatment, 5 min). ( D , E ) Diffusion coefficients for extrasynaptic GluN2A-containing NMDAR and GluN2B-containing NMDARs in control and after cholesterol depletion. ( F , G ) Diffusion coefficients for the mobile fraction of synaptic GluN2A and GluN2B-containing NMDARs in control and after cholesterol depletion. ( H , I ) Fraction of synaptic immobile receptors in control and after cholesterol depletion. (* p

    Article Snippet: Immunocytochemistry Neurons were stained in non-permeabilized conditions with primary antibodies against extracellular epitopes of GluN2A, GluN2B or GluA1 (ACG-002 1:500, Alomone Labs , , ACG-003 1:500, Alomone Labs , , and PC246 1:1000, MERCK ) and then they were depleted of cholesterol.

    Techniques: Diffusion-based Assay

    Distribution of GluN2B immunoreactivity in young and aged GIN mice hippocampus. Panoramic confocal plane showing the distribution of O-LM cells (green) and GluN2B immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN2B, in strata oriens , and pyramidale. Note the presence of GluN2B + clusters in pyramidal neurons in 16-month-old (B3) , but not in 3-month-old (A3) mice. Scale bar: 150 μm for panels (A1,B1 ), 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

    doi: 10.3389/fnagi.2021.782737

    Figure Lengend Snippet: Distribution of GluN2B immunoreactivity in young and aged GIN mice hippocampus. Panoramic confocal plane showing the distribution of O-LM cells (green) and GluN2B immunoreactivity (red) in the hippocampus of 3-month-old (A1) and 16-month-old (B1) mice. Different regions and strata are indicated with dotted lines. (A2,B2) High magnification view from the different CA1 strata in 3-month-old (A2) and 16-month-old (B2) mice. (A3,B3) Enlarged view of the squared regions in panels (A2,B2) , showing double immunofluorescence for GFP/GluN2B, in strata oriens , and pyramidale. Note the presence of GluN2B + clusters in pyramidal neurons in 16-month-old (B3) , but not in 3-month-old (A3) mice. Scale bar: 150 μm for panels (A1,B1 ), 67 μm for panels (A2,B2) , and 21 μm for panels (A3,B3) .

    Article Snippet: Controls were performed omitting the anti-GluN1 or anti-GluN2B antibody, as well as incubating with these antibodies previously pre-absorbed overnight with an excess of its immunogenic peptide (GluN1 blocking peptide, Alomone, Jerusalem, Israel) or (GluN2B blocking peptide, Alomone, Jerusalem, Israel), respectively.

    Techniques: Mouse Assay, Immunofluorescence

    Analysis of the density and percentage of area covered with GluN2B immunoreactive puncta in the somata and in the periphery of O-LM cells during aging. (A–F) Double GFP/GluN2B immunohistochemistry in 3-month-old (A) , 9-month-old (B) , 16-month-old (C) female mice and in 3-month-old (D) , 9-month-old (E) , 16-month-old (F) male mice. In panels (C2,F2) , a detailed view of the GluN2B clustering in aged mice can be observed. (G–I) Graphs showing the density and percentage of area covered with GluN2B immunoreactive puncta in the somata (G1,G2–I2) and in its periphery (G3,G4–I4) in animals segregated by sex (G1–4) , pooled females (H1–4) and males (I1–4) (all graphs represent mean ± SEM., * p -value

    Journal: Frontiers in Aging Neuroscience

    Article Title: Effects of Aging on the Structure and Expression of NMDA Receptors of Somatostatin Expressing Neurons in the Mouse Hippocampus

    doi: 10.3389/fnagi.2021.782737

    Figure Lengend Snippet: Analysis of the density and percentage of area covered with GluN2B immunoreactive puncta in the somata and in the periphery of O-LM cells during aging. (A–F) Double GFP/GluN2B immunohistochemistry in 3-month-old (A) , 9-month-old (B) , 16-month-old (C) female mice and in 3-month-old (D) , 9-month-old (E) , 16-month-old (F) male mice. In panels (C2,F2) , a detailed view of the GluN2B clustering in aged mice can be observed. (G–I) Graphs showing the density and percentage of area covered with GluN2B immunoreactive puncta in the somata (G1,G2–I2) and in its periphery (G3,G4–I4) in animals segregated by sex (G1–4) , pooled females (H1–4) and males (I1–4) (all graphs represent mean ± SEM., * p -value

    Article Snippet: Controls were performed omitting the anti-GluN1 or anti-GluN2B antibody, as well as incubating with these antibodies previously pre-absorbed overnight with an excess of its immunogenic peptide (GluN1 blocking peptide, Alomone, Jerusalem, Israel) or (GluN2B blocking peptide, Alomone, Jerusalem, Israel), respectively.

    Techniques: Immunohistochemistry, Mouse Assay