rabbit polyclonal nr2b antibody  (Alomone Labs)


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    Alomone Labs rabbit polyclonal nr2b antibody
    (A) Representative images of clustering of <t>NR2B</t> in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).
    Rabbit Polyclonal Nr2b Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal nr2b antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal nr2b antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons"

    Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046012

    (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).
    Figure Legend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

    Techniques Used:

    (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.
    Figure Legend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

    Techniques Used: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

    (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.
    Figure Legend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

    Techniques Used: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

    rabbit polyclonal nr2b antibody  (Alomone Labs)


    Bioz Verified Symbol Alomone Labs is a verified supplier
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    Alomone Labs rabbit polyclonal nr2b antibody
    (A) Representative images of clustering of <t>NR2B</t> in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).
    Rabbit Polyclonal Nr2b Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit polyclonal nr2b antibody/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit polyclonal nr2b antibody - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons"

    Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0046012

    (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).
    Figure Legend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

    Techniques Used:

    (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.
    Figure Legend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

    Techniques Used: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

    (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.
    Figure Legend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

    Techniques Used: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

    rabbit anti nr2b  (Alomone Labs)


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    Alomone Labs rabbit anti nr2b
    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or <t>anti-NR2B</t> (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
    Rabbit Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti nr2b/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit anti nr2b - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2"

    Article Title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

    Journal: bioRxiv

    doi: 10.1101/2021.09.30.462507

    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
    Figure Legend Snippet: A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Techniques Used: Immunofluorescence, Staining, Marker, Microscopy, Software, MANN-WHITNEY, In Vitro, Isolation

    anti nr2b  (Alomone Labs)


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    Alomone Labs anti nr2b
    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or <t>anti-NR2B</t> (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
    Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nr2b/product/Alomone Labs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nr2b - by Bioz Stars, 2023-02
    94/100 stars

    Images

    1) Product Images from "Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2"

    Article Title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

    Journal: bioRxiv

    doi: 10.1101/2021.09.30.462507

    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
    Figure Legend Snippet: A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Techniques Used: Immunofluorescence, Staining, Marker, Microscopy, Software, MANN-WHITNEY, In Vitro, Isolation

    anti rabbit alexa 568  (Alomone Labs)


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    Alomone Labs anti rabbit alexa 568
    Anti Rabbit Alexa 568, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    polyclonal rabbit anti glun2b subunit  (Alomone Labs)


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    Alomone Labs polyclonal rabbit anti glun2b subunit
    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Polyclonal Rabbit Anti Glun2b Subunit, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis"

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    Journal: Translational Psychiatry

    doi: 10.1038/s41398-021-01549-7

    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of GluN2B-NMDAR was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Figure Legend Snippet: a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of GluN2B-NMDAR was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).

    Techniques Used: Cell Culture, Diffusion-based Assay, Standard Deviation

    a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). c Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from IIL1RAPL1 wild-type or KO mice. Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in WT and KO conditions (WT, n = 1430 trajectories; KO, n = 1097; p > 0.05).
    Figure Legend Snippet: a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). c Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from IIL1RAPL1 wild-type or KO mice. Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in WT and KO conditions (WT, n = 1430 trajectories; KO, n = 1097; p > 0.05).

    Techniques Used: Transfection, Diffusion-based Assay

    a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p < 0.01, Mann–Whitney test). Data are represented as mean ± sem. c Surface immunostaining of GluN2B-NMDAR onto neurons from control or MAM-exposed pups (synapses were detected with Homer1c-GFP). Scale bars = 5/1 µm (left/right). d Comparison of the GluN2B, GluN2A, GluN3A-NMDAR cluster areas between conditions (GluN2B synaptic cont, n = 2144 clusters; GluN2B synaptic MAM, n = 2468, *** p < 0.001, Student’s t -test; GluN2B extrasynaptic cont, n = 1733; GluN2B extrasynaptic MAM, n = 1421, ** p < 0.01, Student’s t -test; GluN2A cont, n = 56; GluN2A MAM, n = 41; GluN3A cont, n = 59; GluN3A MAM, n = 48).
    Figure Legend Snippet: a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p < 0.01, Mann–Whitney test). Data are represented as mean ± sem. c Surface immunostaining of GluN2B-NMDAR onto neurons from control or MAM-exposed pups (synapses were detected with Homer1c-GFP). Scale bars = 5/1 µm (left/right). d Comparison of the GluN2B, GluN2A, GluN3A-NMDAR cluster areas between conditions (GluN2B synaptic cont, n = 2144 clusters; GluN2B synaptic MAM, n = 2468, *** p < 0.001, Student’s t -test; GluN2B extrasynaptic cont, n = 1733; GluN2B extrasynaptic MAM, n = 1421, ** p < 0.01, Student’s t -test; GluN2A cont, n = 56; GluN2A MAM, n = 41; GluN3A cont, n = 59; GluN3A MAM, n = 48).

    Techniques Used: Cell Culture, Staining, In Vitro, Diffusion-based Assay, MANN-WHITNEY, Immunostaining

    a Spontaneous AMPAR and NMDAR-mediated EPSCs recorded in whole-cell configuration at −70 and +40 mV, respectively, in the CA1 of hippocampal slices from control and MAM-exposed animals. Insets show superimposed examples of spontaneous AMPAR and NMDAR-mediated responses as well as evoked NMDAR-mediated currents. b Spontaneous ( n (cont/MAM) = 14/20; p < 0.05) as well as evoked ( n (cont/MAM) = 16/16; p < 0.05) NMDAR-mediated currents are significantly larger in MAM-exposed animals. Also, the Tau decay was increased by ~25% ( n (cont/MAM) = 8/16; p < 0.05), which is consistent with an increased synaptic expression of GluN2B. c Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. These tonic NMDA currents were significantly decreased in MAM-exposed animals ( n (cont/MAM) = 10/10; p < 0.05), as revealed by a much smaller decrease in holding current with APV. d Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal cell layer after stimulation of hippocampal Schaffer collaterals. Active input displays a significant postsynaptic potentiation (LTP) following high-frequency stimulation at P10 (control), not visible in non-stimulated input ( n = 6; p < 0.05). e When comparing the response to high-frequency stimulation in control and MAM-exposed animals there was a clear deficit in the magnitude of the LTP in MAM-exposed rats between P8 and P12. This deficit was transient and disappeared with age such that the LTP recovered in older animals.
    Figure Legend Snippet: a Spontaneous AMPAR and NMDAR-mediated EPSCs recorded in whole-cell configuration at −70 and +40 mV, respectively, in the CA1 of hippocampal slices from control and MAM-exposed animals. Insets show superimposed examples of spontaneous AMPAR and NMDAR-mediated responses as well as evoked NMDAR-mediated currents. b Spontaneous ( n (cont/MAM) = 14/20; p < 0.05) as well as evoked ( n (cont/MAM) = 16/16; p < 0.05) NMDAR-mediated currents are significantly larger in MAM-exposed animals. Also, the Tau decay was increased by ~25% ( n (cont/MAM) = 8/16; p < 0.05), which is consistent with an increased synaptic expression of GluN2B. c Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. These tonic NMDA currents were significantly decreased in MAM-exposed animals ( n (cont/MAM) = 10/10; p < 0.05), as revealed by a much smaller decrease in holding current with APV. d Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal cell layer after stimulation of hippocampal Schaffer collaterals. Active input displays a significant postsynaptic potentiation (LTP) following high-frequency stimulation at P10 (control), not visible in non-stimulated input ( n = 6; p < 0.05). e When comparing the response to high-frequency stimulation in control and MAM-exposed animals there was a clear deficit in the magnitude of the LTP in MAM-exposed rats between P8 and P12. This deficit was transient and disappeared with age such that the LTP recovered in older animals.

    Techniques Used: Expressing

    a Schematic description of the experimental workflow: control of MAM-exposed pups received intracerebral injections during the second postnatal week of NMDAR surface diffusion modulators. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR on hippocampal neurons exposed to TAT-non sense (TAT-NS, 10 µM), TAT-2B (10 µM), control IgG (10 µg/ml), or IgG direct against extracellular epitope of the GluN2B-NMDAR (10 µg/ml). Scale bars = 500 nm (left/right). c The startle response, i.e., pre-pulse inhibition (PPI), was measured at P60 and compared between conditions ( n = 10–16 rats per group; * P < 0.05, ** P < 0.01, ANOVA 1 followed by Newman–Keuls multiple comparisons test).
    Figure Legend Snippet: a Schematic description of the experimental workflow: control of MAM-exposed pups received intracerebral injections during the second postnatal week of NMDAR surface diffusion modulators. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR on hippocampal neurons exposed to TAT-non sense (TAT-NS, 10 µM), TAT-2B (10 µM), control IgG (10 µg/ml), or IgG direct against extracellular epitope of the GluN2B-NMDAR (10 µg/ml). Scale bars = 500 nm (left/right). c The startle response, i.e., pre-pulse inhibition (PPI), was measured at P60 and compared between conditions ( n = 10–16 rats per group; * P < 0.05, ** P < 0.01, ANOVA 1 followed by Newman–Keuls multiple comparisons test).

    Techniques Used: Diffusion-based Assay, Inhibition

    nr2b subunits  (Alomone Labs)


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    Alomone Labs nr2b subunits
    Nr2b Subunits, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti nr2b  (Alomone Labs)


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    Alomone Labs anti nr2b
    Leptin signaling increases pNR2BY1472 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 pNR2BY1472 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 pNR2BY1472 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 < 0.05, compared to control or wild-type.
    Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Leptin Controls Glutamatergic Synaptogenesis and NMDA-Receptor Trafficking via Fyn Kinase Regulation of NR2B"

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

    Journal: Endocrinology

    doi: 10.1210/endocr/bqz030

    Leptin signaling increases pNR2BY1472 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 pNR2BY1472 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 pNR2BY1472 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 < 0.05, compared to control or wild-type.
    Figure Legend Snippet: Leptin signaling increases pNR2BY1472 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 pNR2BY1472 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 pNR2BY1472 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 < 0.05, compared to control or wild-type.

    Techniques Used: Expressing, Western Blot, Marker, Affinity Purification, Magnetic Beads

    pNR2BY1472 is necessary for leptin-stimulated spine formation. (A) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2BY1472F-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-NR2BY1472F-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; NR2BY1472F: n = 33; NR2BY1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, *P < 0.05, **P < 0.01 compared to control.
    Figure Legend Snippet: pNR2BY1472 is necessary for leptin-stimulated spine formation. (A) Representative fluorescent images of hippocampal neurons expressing Clover and EGFP-NR2B-V5 or EGFP-NR2BY1472F-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-NR2BY1472F-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; NR2BY1472F: n = 33; NR2BY1472F + leptin: n = 33). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, *P < 0.05, **P < 0.01 compared to control.

    Techniques Used: Expressing, Transfection

    Leptin-regulated NR2BY1472 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 pNR2BY1472 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 < 0.05, **P < 0.01.
    Figure Legend Snippet: Leptin-regulated NR2BY1472 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 pNR2BY1472 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 < 0.05, **P < 0.01.

    Techniques Used: 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 < 0.05 compared to control.
    Figure Legend 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 < 0.05 compared to control.

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

    nr2b  (Alomone Labs)


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    Alomone Labs nr2b
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    rabbit polyclonal anti nr2b  (Alomone Labs)


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    Alomone Labs rabbit polyclonal anti nr2b
    Rabbit Polyclonal Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti nmda receptor 2b glun2b  (Alomone Labs)


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    Alomone Labs anti nmda receptor 2b glun2b
    Anti Nmda Receptor 2b Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs rabbit polyclonal nr2b antibody
    (A) Representative images of clustering of <t>NR2B</t> in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).
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    Alomone Labs rabbit anti nr2b
    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or <t>anti-NR2B</t> (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
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    Alomone Labs anti nr2b
    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or <t>anti-NR2B</t> (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
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    Alomone Labs anti rabbit alexa 568
    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or <t>anti-NR2B</t> (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).
    Anti Rabbit Alexa 568, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs polyclonal rabbit anti glun2b subunit
    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Polyclonal Rabbit Anti Glun2b Subunit, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Alomone Labs nr2b subunits
    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Nr2b Subunits, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
    Rabbit Polyclonal Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of <t>GluN2B-NMDAR</t> was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).
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    Image Search Results


    (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

    Journal: PLoS ONE

    Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

    doi: 10.1371/journal.pone.0046012

    Figure Lengend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

    Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

    Techniques:

    (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

    Journal: PLoS ONE

    Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

    doi: 10.1371/journal.pone.0046012

    Figure Lengend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

    Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

    Techniques: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

    (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

    Journal: PLoS ONE

    Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

    doi: 10.1371/journal.pone.0046012

    Figure Lengend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

    Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

    Techniques: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Journal: bioRxiv

    Article Title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

    doi: 10.1101/2021.09.30.462507

    Figure Lengend Snippet: A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Article Snippet: Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v) and immunofluorescence staining was performed using the following antibodies: mouse monoclonal anti-TG2 (IA12 – Tim Johnson, University of Sheffield ( )), guinea pig anti-VGLUT1 (Synaptic System, Goettingen, Germany), rabbit anti-GFAP (Dako, Agilent, Santa Clara, CA, USA), rabbit anti-Shank2 (Synaptic System), rabbit anti-Fibronectin (Sigma-Aldrich), rabbit anti-β-tubulin (Sigma-Aldrich) and rabbit anti-NR2B (Alomone, Jerusalem, Israel).

    Techniques: Immunofluorescence, Staining, Marker, Microscopy, Software, MANN-WHITNEY, In Vitro, Isolation

    A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Journal: bioRxiv

    Article Title: Astrocytic extracellular vesicles modulate neuronal calcium homeostasis via transglutaminase-2

    doi: 10.1101/2021.09.30.462507

    Figure Lengend Snippet: A) Immunofluorescence staining of primary astrocytes. Cells were fixed in 4% paraformaldehyde - 4 % sucrose (w/v), permeabilized (left panels) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), DAPI (blue) and astrocytic marker anti-GFAP (red). Coverslips were visualised by laser scanning Leica SP5 confocal microscope using 63X oil immersion objective. Successive serial optical sections (1 µm) were recorded over 8 µm planes. Scale bar 20 µm. TG2 intensity was calculated by Leica software, divided by number of nuclei, and normalised to permeabilized values. Data is expressed as mean ± SD (N=3, Mann-Whitney test: p=NS). B) Neurons at 12 DIV were fixed and permeabilized (left panel) or non-permeabilized (right panel) and stained with anti-TG2 IA12 (green), anti-β-TUB or anti-NR2B (red) antibodies and DAPI (blue). Scale bar 10 µm. TG2 intensity was calculated as described in A (N=3, Mann-Whitney test: *p<0.05). C) Neurons at 22 Days In Vitro (DIV) were fixed, permeabilized and stained with anti-TG2 IA12 (green), anti-SHANK2 (red) and anti-VGLUT1 (blue) antibodies (N=3). Coverslips were visualised as described in A. Scale bar 20 µm (low magnification) and 5 µm (high magnification). D) Crude synaptosomes were isolated from adult mouse brain and probed with pre- and post-synaptic markers and for astrocyte marker GFAP. B-tubulin was used as loading control. TG2 is enriched in the synaptic fraction (Syn) compared to Low Speed Supernatant (LSS) representing the total lysate. Data is expressed as mean ± SD (N=4 independent experiments, Mann-Whitney test: *p<0.05).

    Article Snippet: The following antibodies were used: mouse monoclonal anti-TG2 (IA12), anti-FLOT-2 (BD Biosciences, Wokingham, United Kingdom), anti-PSD-95 (Neuromab, Davis, C, USA), anti-TOM20 (Santa Cruz, Dallas, Texas, USA) and polyclonal rabbit anti-TG2 (ab421) (Abcam), anti-β-tubulin (Abcam), anti-ALIX (Covalab, Bron, France), anti-NR2B (Alomone, Jerusalem, Israel), anti-VGAT (Synaptic systems, Goettingen, Germany), anti-VGLUT1 (Synaptic systems), anti-GFAP (GeneTex).

    Techniques: Immunofluorescence, Staining, Marker, Microscopy, Software, MANN-WHITNEY, In Vitro, Isolation

    a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of GluN2B-NMDAR was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).

    Journal: Translational Psychiatry

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    doi: 10.1038/s41398-021-01549-7

    Figure Lengend Snippet: a Schematic description of the experimental workflow. CSF from patients with schizophrenic spectrum disorders (SCZSD, n = 12), affective spectrum disorders (AffectSD, n = 9) subarachnoid hemorrhage (SAH, n = 9 patients), brain polytrauma ( n = 3), hemophagocytic lymphohistiocytosis (HLH, n = 1), were collected. Then, single nanoparticle (QDot) tracking of GluN2B-NMDAR was performed onto cultured hippocampal neurons exposed to CSF. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes in dendritic spines of neurons exposed to various CSFs. Scale bar = 300 nm. c Diffusion coefficient (µm 2 /s) of synaptic GluN2B-NMDAR values after exposure for 15–30 min to patients’ CSF. Each plotted dot corresponds to the median diffusion coefficient value of one patient CSF. The mean and standard deviation (SD) are represented for each condition. Right panel , Comparison of GluN2B-NMDAR synaptic diffusion coefficient values after exposure for 15–30 min to patients’ CSF (Control, n = 2757 trajectories; SCZSD, n = 5849; AffectSD, n = 2410; Polytrauma, n = 825; SAB, n = 3957; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). Data are represented as mean ± sem. d Cumulative distributions of GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in control condition and SCZSD CSF ( p < 0.0001, Kolmogorov–Smirnov test).

    Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

    Techniques: Cell Culture, Diffusion-based Assay, Standard Deviation

    a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). c Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from IIL1RAPL1 wild-type or KO mice. Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in WT and KO conditions (WT, n = 1430 trajectories; KO, n = 1097; p > 0.05).

    Journal: Translational Psychiatry

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    doi: 10.1038/s41398-021-01549-7

    Figure Lengend Snippet: a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p < 0.0001, Kruskal–Wallis followed by Dunn’s multiple comparison test). c Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from IIL1RAPL1 wild-type or KO mice. Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in WT and KO conditions (WT, n = 1430 trajectories; KO, n = 1097; p > 0.05).

    Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

    Techniques: Transfection, Diffusion-based Assay

    a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p < 0.01, Mann–Whitney test). Data are represented as mean ± sem. c Surface immunostaining of GluN2B-NMDAR onto neurons from control or MAM-exposed pups (synapses were detected with Homer1c-GFP). Scale bars = 5/1 µm (left/right). d Comparison of the GluN2B, GluN2A, GluN3A-NMDAR cluster areas between conditions (GluN2B synaptic cont, n = 2144 clusters; GluN2B synaptic MAM, n = 2468, *** p < 0.001, Student’s t -test; GluN2B extrasynaptic cont, n = 1733; GluN2B extrasynaptic MAM, n = 1421, ** p < 0.01, Student’s t -test; GluN2A cont, n = 56; GluN2A MAM, n = 41; GluN3A cont, n = 59; GluN3A MAM, n = 48).

    Journal: Translational Psychiatry

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    doi: 10.1038/s41398-021-01549-7

    Figure Lengend Snippet: a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p < 0.01, Mann–Whitney test). Data are represented as mean ± sem. c Surface immunostaining of GluN2B-NMDAR onto neurons from control or MAM-exposed pups (synapses were detected with Homer1c-GFP). Scale bars = 5/1 µm (left/right). d Comparison of the GluN2B, GluN2A, GluN3A-NMDAR cluster areas between conditions (GluN2B synaptic cont, n = 2144 clusters; GluN2B synaptic MAM, n = 2468, *** p < 0.001, Student’s t -test; GluN2B extrasynaptic cont, n = 1733; GluN2B extrasynaptic MAM, n = 1421, ** p < 0.01, Student’s t -test; GluN2A cont, n = 56; GluN2A MAM, n = 41; GluN3A cont, n = 59; GluN3A MAM, n = 48).

    Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

    Techniques: Cell Culture, Staining, In Vitro, Diffusion-based Assay, MANN-WHITNEY, Immunostaining

    a Spontaneous AMPAR and NMDAR-mediated EPSCs recorded in whole-cell configuration at −70 and +40 mV, respectively, in the CA1 of hippocampal slices from control and MAM-exposed animals. Insets show superimposed examples of spontaneous AMPAR and NMDAR-mediated responses as well as evoked NMDAR-mediated currents. b Spontaneous ( n (cont/MAM) = 14/20; p < 0.05) as well as evoked ( n (cont/MAM) = 16/16; p < 0.05) NMDAR-mediated currents are significantly larger in MAM-exposed animals. Also, the Tau decay was increased by ~25% ( n (cont/MAM) = 8/16; p < 0.05), which is consistent with an increased synaptic expression of GluN2B. c Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. These tonic NMDA currents were significantly decreased in MAM-exposed animals ( n (cont/MAM) = 10/10; p < 0.05), as revealed by a much smaller decrease in holding current with APV. d Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal cell layer after stimulation of hippocampal Schaffer collaterals. Active input displays a significant postsynaptic potentiation (LTP) following high-frequency stimulation at P10 (control), not visible in non-stimulated input ( n = 6; p < 0.05). e When comparing the response to high-frequency stimulation in control and MAM-exposed animals there was a clear deficit in the magnitude of the LTP in MAM-exposed rats between P8 and P12. This deficit was transient and disappeared with age such that the LTP recovered in older animals.

    Journal: Translational Psychiatry

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    doi: 10.1038/s41398-021-01549-7

    Figure Lengend Snippet: a Spontaneous AMPAR and NMDAR-mediated EPSCs recorded in whole-cell configuration at −70 and +40 mV, respectively, in the CA1 of hippocampal slices from control and MAM-exposed animals. Insets show superimposed examples of spontaneous AMPAR and NMDAR-mediated responses as well as evoked NMDAR-mediated currents. b Spontaneous ( n (cont/MAM) = 14/20; p < 0.05) as well as evoked ( n (cont/MAM) = 16/16; p < 0.05) NMDAR-mediated currents are significantly larger in MAM-exposed animals. Also, the Tau decay was increased by ~25% ( n (cont/MAM) = 8/16; p < 0.05), which is consistent with an increased synaptic expression of GluN2B. c Tonic NMDA currents were recorded as the subtracted holding current at a depolarized potential of +40 mV before and after the addition of APV. These tonic NMDA currents were significantly decreased in MAM-exposed animals ( n (cont/MAM) = 10/10; p < 0.05), as revealed by a much smaller decrease in holding current with APV. d Field excitatory postsynaptic potential (fEPSP) recorded in CA1 pyramidal cell layer after stimulation of hippocampal Schaffer collaterals. Active input displays a significant postsynaptic potentiation (LTP) following high-frequency stimulation at P10 (control), not visible in non-stimulated input ( n = 6; p < 0.05). e When comparing the response to high-frequency stimulation in control and MAM-exposed animals there was a clear deficit in the magnitude of the LTP in MAM-exposed rats between P8 and P12. This deficit was transient and disappeared with age such that the LTP recovered in older animals.

    Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

    Techniques: Expressing

    a Schematic description of the experimental workflow: control of MAM-exposed pups received intracerebral injections during the second postnatal week of NMDAR surface diffusion modulators. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR on hippocampal neurons exposed to TAT-non sense (TAT-NS, 10 µM), TAT-2B (10 µM), control IgG (10 µg/ml), or IgG direct against extracellular epitope of the GluN2B-NMDAR (10 µg/ml). Scale bars = 500 nm (left/right). c The startle response, i.e., pre-pulse inhibition (PPI), was measured at P60 and compared between conditions ( n = 10–16 rats per group; * P < 0.05, ** P < 0.01, ANOVA 1 followed by Newman–Keuls multiple comparisons test).

    Journal: Translational Psychiatry

    Article Title: Alteration of NMDA receptor trafficking as a cellular hallmark of psychosis

    doi: 10.1038/s41398-021-01549-7

    Figure Lengend Snippet: a Schematic description of the experimental workflow: control of MAM-exposed pups received intracerebral injections during the second postnatal week of NMDAR surface diffusion modulators. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR on hippocampal neurons exposed to TAT-non sense (TAT-NS, 10 µM), TAT-2B (10 µM), control IgG (10 µg/ml), or IgG direct against extracellular epitope of the GluN2B-NMDAR (10 µg/ml). Scale bars = 500 nm (left/right). c The startle response, i.e., pre-pulse inhibition (PPI), was measured at P60 and compared between conditions ( n = 10–16 rats per group; * P < 0.05, ** P < 0.01, ANOVA 1 followed by Newman–Keuls multiple comparisons test).

    Article Snippet: For x-link experiments, the control group received 1 µl of anti-rabbit alexa 568 (control IgG, 1/5), while the GluN2B-x-link group received 1 mg of polyclonal rabbit anti-GluN2B subunit (Alomone Labs).

    Techniques: Diffusion-based Assay, Inhibition