glun2b  (Alomone Labs)


Bioz Verified Symbol Alomone Labs is a verified supplier
Bioz Manufacturer Symbol Alomone Labs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95

    Structured Review

    Alomone Labs glun2b
    Cholesterol depletion reduces synaptic localization of NMDARs. ( A , C ) Colocalization of surface GluN2A (A, green) or <t>GluN2B</t> (C, green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (10 mM MβCD pretreatment, 5 min). Scale bar 2 µm. ( B , D ) Bar graphs showing Pearson's coefficient for the colocalization indicate the reduction of synaptic localization of GluN2A and GluN2B after cholesterol depletion. ( E ) Colocalization of surface GluA1 (green) and the postsynaptic marker Shank (red) in control and cholesterol-depleted neurons (MβCD). Scale bar 2 µm. ( F ) Bar graph showing Pearson's coefficient for the colocalization. ( G ) Examples of typical dual AMPAR-NMDAR mEPSCs in control autaptic neurons having various AMPAR to NMDAR ratio. ( H ) Examples of typical dual AMPAR-NMDAR mEPSCs in 10 mM MβCD-pretreated autaptic neurons. ( I ) Examples of NMDAR mEPSCs obtained from average dual mEPSCs after AMPAR mEPSC subtraction. A control neuron (top trace) and a cholesterol-depleted neuron (bottom trace). The arrows indicate mEPSC onsets. ( J ) The comparison of average amplitude of NMDAR mEPSCs in control neurons and in cholesterol-depleted neurons. (* p
    Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/glun2b/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    glun2b - by Bioz Stars, 2022-07
    95/100 stars

    Images

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

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

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-69454-5

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

    Techniques Used: Marker

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

    Techniques Used: Diffusion-based Assay

    2) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00313

    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P
    Figure Legend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P
    Figure Legend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Techniques Used: Western Blot

    3) Product Images from "Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses"

    Article Title: Surface dynamics of GluN2B-NMDA receptors controls plasticity of maturing glutamate synapses

    Journal: The EMBO Journal

    doi: 10.1002/embj.201386356

    Regulation of GluN2B-NMDAR surface dynamics by CAMKII and CKII activities Representative trajectories (25-s duration, 20-Hz acquisition) of surface QD-conjugated GluN2B-NMDAR within Homer 1c-GFP-labeled synaptic areas (gray) before and after chem LTP in immature hippocampal neuron (9–12 div) in control (green), TBB (orange), and KN62 (red) conditions. Scale bar = 1 μm. Synaptic GluN2B-NMDAR surface diffusion change (normalized to baseline for each condition) after chem LTP in immature neurons (
    Figure Legend Snippet: Regulation of GluN2B-NMDAR surface dynamics by CAMKII and CKII activities Representative trajectories (25-s duration, 20-Hz acquisition) of surface QD-conjugated GluN2B-NMDAR within Homer 1c-GFP-labeled synaptic areas (gray) before and after chem LTP in immature hippocampal neuron (9–12 div) in control (green), TBB (orange), and KN62 (red) conditions. Scale bar = 1 μm. Synaptic GluN2B-NMDAR surface diffusion change (normalized to baseline for each condition) after chem LTP in immature neurons (

    Techniques Used: Labeling, Diffusion-based Assay

    Antibodies against extracellular epitopes of NMDAR from autoimmune encephalitis patients acutely prevent chem LTP Schematic diagram of the anti-NMDAR IgG isolation procedure from anti-NMDAR encephalitis patients. The cerebrospinal fluid (CSF) was collected and IgG were purified for in vitro imaging experiments. Lower panels: note the high co-localization of surface staining from surface patient anti-NMDAR IgG (“sPat. IgG,” green) and commercial anti-GluN1 antibodies (“sGluN1,” red). Scale bar = 1 μm. Representative GluN2B-NMDAR-QD trajectories from neurons incubated either with control or with patient IgG. Note the massive reduction in surface dynamics. Scale bar = 250 nm. Representative images of hippocampal neurons in the basal conditions or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Neurons were incubated either with no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG). Scale bar = 20 μm. Right panel: Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons in no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG) conditions. Hippocampal neurons expressing either GluN1-SEP or GluA1-SEP were incubated with IgG (5 μg/ml) either from control or from anti-NMDAR patients for 20–25 min. Note that patient IgG do not affect GluN1-SEP distribution. Neurons were stimulated with a chem LTP protocol and each synaptic GluA1-AMPAR cluster was followed over time. Note that chem LTP increased the intensity of GluA1-SEP in synaptic clusters (arrows) only in control IgG condition. Scale bars = 1 μm. Lower panels: Quantification of the GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses in control or patient IgG conditions. For each neuron, GluA1 synaptic fluorescence intensity was quantified before and 10–15 min after chem LTP. The GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses significantly increased in control condition ( n = 6 neurons; Student's t -test, * P
    Figure Legend Snippet: Antibodies against extracellular epitopes of NMDAR from autoimmune encephalitis patients acutely prevent chem LTP Schematic diagram of the anti-NMDAR IgG isolation procedure from anti-NMDAR encephalitis patients. The cerebrospinal fluid (CSF) was collected and IgG were purified for in vitro imaging experiments. Lower panels: note the high co-localization of surface staining from surface patient anti-NMDAR IgG (“sPat. IgG,” green) and commercial anti-GluN1 antibodies (“sGluN1,” red). Scale bar = 1 μm. Representative GluN2B-NMDAR-QD trajectories from neurons incubated either with control or with patient IgG. Note the massive reduction in surface dynamics. Scale bar = 250 nm. Representative images of hippocampal neurons in the basal conditions or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Neurons were incubated either with no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG). Scale bar = 20 μm. Right panel: Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons in no IgG, controls' IgG (Cont. IgG), or patients' IgG (Pat. IgG) conditions. Hippocampal neurons expressing either GluN1-SEP or GluA1-SEP were incubated with IgG (5 μg/ml) either from control or from anti-NMDAR patients for 20–25 min. Note that patient IgG do not affect GluN1-SEP distribution. Neurons were stimulated with a chem LTP protocol and each synaptic GluA1-AMPAR cluster was followed over time. Note that chem LTP increased the intensity of GluA1-SEP in synaptic clusters (arrows) only in control IgG condition. Scale bars = 1 μm. Lower panels: Quantification of the GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses in control or patient IgG conditions. For each neuron, GluA1 synaptic fluorescence intensity was quantified before and 10–15 min after chem LTP. The GluA1-AMPAR synaptic content and percentage of potentiated GluA1-AMPAR synapses significantly increased in control condition ( n = 6 neurons; Student's t -test, * P

    Techniques Used: Isolation, Purification, In Vitro, Imaging, Staining, Incubation, Expressing, Fluorescence

    The activity-dependent shift in CaMKII dynamics within dendritic spines is regulated by GluN2B-NMDAR dynamics Representative immunoblots showing the immunoprecipitation (IP) of CaMKII (α form) and phospho-CaMKII-Thr286 with GluN2B in membrane fractions from hippocampal slices (P17–20 rats) incubated with buffer (control) or GluN1 x-link. Lower panel: the ratio between CaMKII and GluN2B optical densities is represented ( n = 3 independent experiments). SM, start material; No Ab., no antibody; Cont., control. CaMKII-GFP was detected and imaged in spines before (basal) and after chem LTP in control and GluN1x-link conditions. Scale bar = 1 μm. Lower panel: CaMKII-GFP fluorescence intensity was compared between these conditions (basal: n = 6 neuronal fields, N = 765 spines; chem LTP: n = 11 neuronal fields, N = 1,688 spines; basal versus chem LTP, Student's t -test, *** P
    Figure Legend Snippet: The activity-dependent shift in CaMKII dynamics within dendritic spines is regulated by GluN2B-NMDAR dynamics Representative immunoblots showing the immunoprecipitation (IP) of CaMKII (α form) and phospho-CaMKII-Thr286 with GluN2B in membrane fractions from hippocampal slices (P17–20 rats) incubated with buffer (control) or GluN1 x-link. Lower panel: the ratio between CaMKII and GluN2B optical densities is represented ( n = 3 independent experiments). SM, start material; No Ab., no antibody; Cont., control. CaMKII-GFP was detected and imaged in spines before (basal) and after chem LTP in control and GluN1x-link conditions. Scale bar = 1 μm. Lower panel: CaMKII-GFP fluorescence intensity was compared between these conditions (basal: n = 6 neuronal fields, N = 765 spines; chem LTP: n = 11 neuronal fields, N = 1,688 spines; basal versus chem LTP, Student's t -test, *** P

    Techniques Used: Activity Assay, Western Blot, Immunoprecipitation, Incubation, Fluorescence

    Schematic model of the dynamic interplay between surface GluN2B-NMDAR and CaMKII during synaptic long-term potentiation (LTP) In basal conditions, GluN2B-NMDAR surface diffusion is regulated by the activity of CaMKII. During activity-dependent changes in glutamatergic synaptic transmission, increased surface dynamics of GluN2B-NMDAR favors the intracellular redistribution of CaMKII through their direct interaction, promoting the accumulation of CaMKII in spines.
    Figure Legend Snippet: Schematic model of the dynamic interplay between surface GluN2B-NMDAR and CaMKII during synaptic long-term potentiation (LTP) In basal conditions, GluN2B-NMDAR surface diffusion is regulated by the activity of CaMKII. During activity-dependent changes in glutamatergic synaptic transmission, increased surface dynamics of GluN2B-NMDAR favors the intracellular redistribution of CaMKII through their direct interaction, promoting the accumulation of CaMKII in spines.

    Techniques Used: Diffusion-based Assay, Activity Assay, Transmission Assay

    The surface diffusion of synaptic GluN2B-NMDAR is rapidly increased after chem LTP in immature neurons Upper panels: Representative dendritic fragments of a hippocampal neuron (13 div) expressing GluA1-SEP and Homer 1c-DsRed. Clusters of GluA1-SEP were imaged for at least 30 min. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. GluA1-SEP intensity was measured in synapses (Homer1c cluster) before and after the induction of chem LTP (see Materials and Methods). Note that 10 min after chem LTP, the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. Arrows designate synapses (i.e., Homer). Bottom panels: magnifications and time-lapses of the outlined GluA1-SEP synaptic clusters. Scale bar = 5 μm; insets = 500 nm; bottom panels = 350 nm. Left panel: Representative example of a GluA1-SEP fluorescence time course before and after chem LTP (red) or buffer (black) protocol. The average GluA1-SEP synaptic intensity was significantly increased after chem LTP (24 min after induction) (middle panel). Student's t -test, *** P
    Figure Legend Snippet: The surface diffusion of synaptic GluN2B-NMDAR is rapidly increased after chem LTP in immature neurons Upper panels: Representative dendritic fragments of a hippocampal neuron (13 div) expressing GluA1-SEP and Homer 1c-DsRed. Clusters of GluA1-SEP were imaged for at least 30 min. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. GluA1-SEP intensity was measured in synapses (Homer1c cluster) before and after the induction of chem LTP (see Materials and Methods). Note that 10 min after chem LTP, the GluA1-SEP fluorescence intensity increased in postsynaptic clusters. Arrows designate synapses (i.e., Homer). Bottom panels: magnifications and time-lapses of the outlined GluA1-SEP synaptic clusters. Scale bar = 5 μm; insets = 500 nm; bottom panels = 350 nm. Left panel: Representative example of a GluA1-SEP fluorescence time course before and after chem LTP (red) or buffer (black) protocol. The average GluA1-SEP synaptic intensity was significantly increased after chem LTP (24 min after induction) (middle panel). Student's t -test, *** P

    Techniques Used: Diffusion-based Assay, Expressing, Staining, Fluorescence

    The surface cross-linking of NMDAR blocks chem LTP Comparison of the GluA1-SEP fluorescence within synapses (white dotted circle) in spines from control ( n = 786 synapses), GluN1 x-link ( n = 1324), or GluN2B x-link ( n = 987) condition. The dendritic shaft is indicated by the arrow head. Scale bar = 1 μm. The bar graphs represent the mean value ± s.e.m. Time-lapse imaging of spine areas containing GluA1-SEP (white dotted circle) from immature hippocampal neurons in control (no stimulation), chem LTP, chem LTP + GluN1 x-link, and chem LTP + GluN2B x-link conditions. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. Scale bar = 1 μm. Comparison of the normalized GluA1-SEP fluorescence intensity within synapses in control ( n = 786 synapses), chem LTP alone ( n = 1324, *** P
    Figure Legend Snippet: The surface cross-linking of NMDAR blocks chem LTP Comparison of the GluA1-SEP fluorescence within synapses (white dotted circle) in spines from control ( n = 786 synapses), GluN1 x-link ( n = 1324), or GluN2B x-link ( n = 987) condition. The dendritic shaft is indicated by the arrow head. Scale bar = 1 μm. The bar graphs represent the mean value ± s.e.m. Time-lapse imaging of spine areas containing GluA1-SEP (white dotted circle) from immature hippocampal neurons in control (no stimulation), chem LTP, chem LTP + GluN1 x-link, and chem LTP + GluN2B x-link conditions. The pseudocolor representation shows the different intensity levels of the GluA1-SEP staining. Scale bar = 1 μm. Comparison of the normalized GluA1-SEP fluorescence intensity within synapses in control ( n = 786 synapses), chem LTP alone ( n = 1324, *** P

    Techniques Used: Fluorescence, Imaging, Staining

    Surface cross-linking of NMDAR specifically impairs their surface diffusion without affecting their function Trajectories of single surface QD-GluN1-NMDAR (30-Hz acquisition, 30-s duration) in hippocampal neurons in control (left) and GluN1x-link (right) conditions. A schematic representation of the NMDAR x-link technique using primary anti-GluN (I ary Ab) and secondary (II ary Ab) antibodies is shown in the middle panel. Insets: enlarged GluN1-QD trajectories. Field scale bar = 5 μm; inset scale bar = 1 μm. Cumulative distribution of GluN1-NMDAR instantaneous surface diffusion coefficients in control and GluN1x-link conditions. Note the leftward shift of the curve in the GluN1x-link condition, indicating a slowdown of surface diffusion. Representative FRAP acquisition of GluA1-SEP in control and GluN1-NMDAR x-link conditions. The circles indicate the bleached regions. Scale bar = 5 μm. Average FRAP recovery curves of GluA1-SEP fluorescence in control ( n = 13), GluN1 x-link ( n = 3), and GluN2B x-link ( n = 4) conditions. Full lines represent the average recovery, while dotted lines represent the mean ± s.e.m. The fluorescence recovery of surface synaptic GluA1-SEP remained unaffected in all conditions ( P > 0.05). Representative images of a hippocampal neuron in the basal condition or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Scale bar = 20 μm. Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons ( n = 29). Relative comparison (percent of basal) of a transient calcium rise induced by glutamate in control ( n = 29), control + AP5 ( n = 12, Student's t -test, ** P
    Figure Legend Snippet: Surface cross-linking of NMDAR specifically impairs their surface diffusion without affecting their function Trajectories of single surface QD-GluN1-NMDAR (30-Hz acquisition, 30-s duration) in hippocampal neurons in control (left) and GluN1x-link (right) conditions. A schematic representation of the NMDAR x-link technique using primary anti-GluN (I ary Ab) and secondary (II ary Ab) antibodies is shown in the middle panel. Insets: enlarged GluN1-QD trajectories. Field scale bar = 5 μm; inset scale bar = 1 μm. Cumulative distribution of GluN1-NMDAR instantaneous surface diffusion coefficients in control and GluN1x-link conditions. Note the leftward shift of the curve in the GluN1x-link condition, indicating a slowdown of surface diffusion. Representative FRAP acquisition of GluA1-SEP in control and GluN1-NMDAR x-link conditions. The circles indicate the bleached regions. Scale bar = 5 μm. Average FRAP recovery curves of GluA1-SEP fluorescence in control ( n = 13), GluN1 x-link ( n = 3), and GluN2B x-link ( n = 4) conditions. Full lines represent the average recovery, while dotted lines represent the mean ± s.e.m. The fluorescence recovery of surface synaptic GluA1-SEP remained unaffected in all conditions ( P > 0.05). Representative images of a hippocampal neuron in the basal condition or after glutamate (30 μM) application. The pseudocolor representation shows the different intensity levels of the calcium indicator (Fluo4-AM, 2 μM) before and after the glutamate stimulation. Scale bar = 20 μm. Average calcium intensity change (ΔF/F0) over time after glutamate stimulation of hippocampal neurons ( n = 29). Relative comparison (percent of basal) of a transient calcium rise induced by glutamate in control ( n = 29), control + AP5 ( n = 12, Student's t -test, ** P

    Techniques Used: Diffusion-based Assay, Fluorescence

    Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P
    Figure Legend Snippet: Synaptic GluN2B-NMDAR are laterally redistributed in the synaptic area following chem LTP in immature neurons Detection of a single QD (30-Hz acquisition) in our experimental conditions. The high signal-to-noise ratio (SNR) ( > 5) enables the detection and location of the signal with a high pointing accuracy (˜30 nm). The QD fluorescence is quantified on a pseudocolor scale (low: red; high: yellow). Scale bar = 800 nm. A 500-frame stack is obtained while tracking down a single NMDAR/QD complex. On each frame, a single GluN2B- (green) or GluN2A- (blue) QD particle complex is detected and precisely located within synaptic (dark gray) and perisynaptic (320-nm annulus around the synapse; light gray) areas. Those 500 locations are then projected on a single image, providing the successive positions of this receptor/particle complex during the 500-frame stack. Note that GluN2A-NMDAR are more concentrated within the core of the PSD. Scale bar image = 300 nm; synaptic areas = 200 nm. Relative fraction of synaptic and perisynaptic GluN2-QD particles, calculated before and after chem LTP for both GluN2B- (left) and GluN2A-NMDAR (right) ( n = 25 and 20 dendritic fields before and after chem LTP, respectively). Note the significant decrease in the relative synaptic content in GluN2B-NMDAR particles right after chem LTP (Student's t -test, ** P

    Techniques Used: Fluorescence

    4) Product Images from "Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition"

    Article Title: Lack of the Actin Capping Protein, Eps8, Affects NMDA-Type Glutamate Receptor Function and Composition

    Journal: Frontiers in Molecular Neuroscience

    doi: 10.3389/fnmol.2018.00313

    Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P
    Figure Legend Snippet: Increased GluN2B and reduced GluN2A synaptic content of NMDARs in Eps8 KO neurons. (A) Representative traces of NMDAR mediated mEPSCs recorded from WT and EPS8 KO hippocampal neurons in the presence or not of the GluN1/GluN2B blocker Ifenprodil (3 μM). Scale bar 10 pA, 50 ms. (B,C) Quantification of the inhibitory effect of ifenprodil on the NMDA-mEPSC frequency and amplitude respectively showing increased inhibition in KO neurons with respect to WT (B: Mann Whitney Test * P = 0.0270; C: Unpaired t test ** P = 0.0065). (D) Examples of whole-cell currents elicited in WT and EPS8 KO hippocampal neurons by application of a saturating concentration of NMDA (200 μm) in the presence or absence of GluN2B-selective antagonist ifenprodil (3 μM). Scale bar 300 pA, 2 s. Quantitation of current density (E) , %age of ifenprodil inhibition (F) showing a larger amount of ifenprodil-dependent inhibition in KO neurons with respect to WT (Mann Whitney Test * P = 0.0127). (G) Summary distribution graph of whole-cell currents elicited in WT and EPS8 KO treated or not with tricine (10 mM) showing a reduced GluN2A-dependent increase of current density upon tricine exposure in KO neurons with respect to WT (Mann Whitney Test, *** P

    Techniques Used: Mass Spectrometry, Inhibition, MANN-WHITNEY, Concentration Assay, Quantitation Assay

    Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P
    Figure Legend Snippet: Increased levels of GluN2B-containing and Y1472-GluN2B phosphorylated NMDARs in synaptic triton insoluble fraction (TIF) from Eps8 KO tissue. (A) Western blotting (WB) analysis confirmed that TIF preparation was actually enriched in postsynaptic proteins. (B–D) WB analysis and relative quantification in homogenate and TIF fractions for the indicated antibodies. All the WB were run twice (data are mean ± SEM, N = 4; Kruskal-Wallis test followed by Dunn’s multiple Comparison test * P

    Techniques Used: Western Blot

    5) Product Images from "Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells"

    Article Title: Functional NMDA receptors are expressed by human pulmonary artery smooth muscle cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-021-87667-0

    Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p
    Figure Legend Snippet: Functional NMDA receptors are localized on the surface of HPASMCs. Immunofluorescence staining was performed on non-permeabilized HPASMCs. Both GluN1 and GluN2 (2B and 2D) subunits were detected on the surface of HPASMCs ( A , B ). GluN1 co-localized with GluN2B ( A ) and GluN2D ( B ) on the surface of HPASMCs, respectively. Results are representative images taken from at least three separate experiments. Scale bar = 100 µM. ( C , D ) are representative scatter plots of each fluorescent pixel from confocal images with GluN1 green fluorescent intensity along the x -axis and GluN2 red fluorescent intensity along the y -axis. The shaded area in the upper right quadrant represents colocalized pixels above background with the associated Pearson correlation coefficient indicated for all colocalized pixels. ( E ) Representative traces for cultured HPASMCs in response to 100 μM NMDA and 10 μM glycine treatment. Cells were clamped at − 50 mV and whole cell recordings were performed. ( F ) NMDA and glycine treatment evoked an inward whole-cell current of 10.9 ± 1.7 pA (Mean ± SE, **p

    Techniques Used: Functional Assay, Immunofluorescence, Staining, Cell Culture

    6) Product Images from "Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy"

    Article Title: Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1006684

    Synaptic expression and surface trafficking of glutamate receptors is affected in Ptpn11 D61Y . (A) Representative examples of the staining of synaptic surface (left) and total (right) glutamate receptors containing GluA1 or GluN2B subunit in neurons from control and from Ptpn11 D61Y . Excitatory synapses were stained for Homer1 and Bassoon. (B, C) Synaptic IF intensity and density (puncta per 20 μm of proximal dendrite) of surface (B) and total (C) staining for all tested receptor subunits are shown. Values are normalized to the respective value in control, represented by the dashed line in the graphs. Note the reduced surface expression of GluA2 and GluN2B and the decrease of the overall expression of GluA1 in Ptpn11 D61Y neurons as compared to controls. All numerical values and statistics are listed in S1 Table . (D, E) The rate of internalization of the GluA1-containing AMPARs is reduced in Ptpn11 D61Y cells. Scale bars: 5 μm. Data are presented as mean ± SEM and analyzed using unpaired t-test (*p≤0.05, **p≤0.01, ***p≤0.001). Numbers in columns indicate the number of cells analyzed.
    Figure Legend Snippet: Synaptic expression and surface trafficking of glutamate receptors is affected in Ptpn11 D61Y . (A) Representative examples of the staining of synaptic surface (left) and total (right) glutamate receptors containing GluA1 or GluN2B subunit in neurons from control and from Ptpn11 D61Y . Excitatory synapses were stained for Homer1 and Bassoon. (B, C) Synaptic IF intensity and density (puncta per 20 μm of proximal dendrite) of surface (B) and total (C) staining for all tested receptor subunits are shown. Values are normalized to the respective value in control, represented by the dashed line in the graphs. Note the reduced surface expression of GluA2 and GluN2B and the decrease of the overall expression of GluA1 in Ptpn11 D61Y neurons as compared to controls. All numerical values and statistics are listed in S1 Table . (D, E) The rate of internalization of the GluA1-containing AMPARs is reduced in Ptpn11 D61Y cells. Scale bars: 5 μm. Data are presented as mean ± SEM and analyzed using unpaired t-test (*p≤0.05, **p≤0.01, ***p≤0.001). Numbers in columns indicate the number of cells analyzed.

    Techniques Used: Expressing, Staining

    7) Product Images from "Selective Impairment of Spatial Cognition Caused by Autoantibodies to the N-Methyl-d-Aspartate Receptor"

    Article Title: Selective Impairment of Spatial Cognition Caused by Autoantibodies to the N-Methyl-d-Aspartate Receptor

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2015.05.027

    DNRAbs bind to NMDARs expressed in the cell membrane. The panels show the binding of G11 (human monoclonal DNRAb cloned from a SLE patient) to transfected HEK-293 T cells. (A) Left , GluN1–GluN2A double transfected cells show clear surface binding of G11 (top, green signal, Alexa 488) but not B1, the control human antibody without NMDAR binding (bottom). Middle , strong binding of rabbit anti-GluN2A antibody to surface-expressed GluN2A (red signal, Alexa 594). Right , merged signal indicates that G11 binds to the GluN2A-containing NMDARs. (B) GluN1–GluN2B double transfected cells show a similar binding pattern for the GluN2B-containing NMDARs. (C) Binding of G11 to rabbit polyclonal antibodies was excluded by demonstrating that G11 does not bind to the cell surface of HEK-293 T cells incubated with rabbit polyclonal GLUT2 antibody, which abundantly binds to the cell surface of HEK-293 T cells (red staining). Bar, 30 μm.
    Figure Legend Snippet: DNRAbs bind to NMDARs expressed in the cell membrane. The panels show the binding of G11 (human monoclonal DNRAb cloned from a SLE patient) to transfected HEK-293 T cells. (A) Left , GluN1–GluN2A double transfected cells show clear surface binding of G11 (top, green signal, Alexa 488) but not B1, the control human antibody without NMDAR binding (bottom). Middle , strong binding of rabbit anti-GluN2A antibody to surface-expressed GluN2A (red signal, Alexa 594). Right , merged signal indicates that G11 binds to the GluN2A-containing NMDARs. (B) GluN1–GluN2B double transfected cells show a similar binding pattern for the GluN2B-containing NMDARs. (C) Binding of G11 to rabbit polyclonal antibodies was excluded by demonstrating that G11 does not bind to the cell surface of HEK-293 T cells incubated with rabbit polyclonal GLUT2 antibody, which abundantly binds to the cell surface of HEK-293 T cells (red staining). Bar, 30 μm.

    Techniques Used: Binding Assay, Clone Assay, Transfection, Incubation, Staining

    8) Product Images from "Immunosuppression by N-Methyl-d-Aspartate Receptor Antagonists Is Mediated through Inhibition of Kv1.3 and KCa3.1 Channels in T Cells"

    Article Title: Immunosuppression by N-Methyl-d-Aspartate Receptor Antagonists Is Mediated through Inhibition of Kv1.3 and KCa3.1 Channels in T Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01273-13

    NMDAR antagonists impair T-cell proliferation. (A) RT-PCR analysis of mRNA expression of NMDAR subunits GluN1, GluN2A, and GluN2B in thymocytes, brain (br.), peripheral CD4 + T cells, as well as CD4 + and CD8 + T cells activated with CD3 and CD28 Abs (3
    Figure Legend Snippet: NMDAR antagonists impair T-cell proliferation. (A) RT-PCR analysis of mRNA expression of NMDAR subunits GluN1, GluN2A, and GluN2B in thymocytes, brain (br.), peripheral CD4 + T cells, as well as CD4 + and CD8 + T cells activated with CD3 and CD28 Abs (3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    9) Product Images from "Immunosuppression by N-Methyl-d-Aspartate Receptor Antagonists Is Mediated through Inhibition of Kv1.3 and KCa3.1 Channels in T Cells"

    Article Title: Immunosuppression by N-Methyl-d-Aspartate Receptor Antagonists Is Mediated through Inhibition of Kv1.3 and KCa3.1 Channels in T Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01273-13

    NMDAR antagonists impair T-cell proliferation. (A) RT-PCR analysis of mRNA expression of NMDAR subunits GluN1, GluN2A, and GluN2B in thymocytes, brain (br.), peripheral CD4 + T cells, as well as CD4 + and CD8 + T cells activated with CD3 and CD28 Abs (3
    Figure Legend Snippet: NMDAR antagonists impair T-cell proliferation. (A) RT-PCR analysis of mRNA expression of NMDAR subunits GluN1, GluN2A, and GluN2B in thymocytes, brain (br.), peripheral CD4 + T cells, as well as CD4 + and CD8 + T cells activated with CD3 and CD28 Abs (3

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Expressing

    10) Product Images from "Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons"

    Article Title: Fingolimod Limits Acute Aβ Neurotoxicity and Promotes Synaptic Versus Extrasynaptic NMDA Receptor Functionality in Hippocampal Neurons

    Journal: Scientific Reports

    doi: 10.1038/srep41734

    FTY720 modulates GLUN2B localization in the postsynaptic fraction and increases surface GLUN2B and GLUN2A expression at synapses. ( A , B ) Western blotting of the homogenate (HOMO), postsynaptic triton insoluble fraction (TIF) and triton soluble fraction (TSF) obtained from control and FTY720-treated hippocampal neurons (DIV14). Tubulin is used as loading control, while PSD-95 as a marker of postsynaptic fraction. FTY720 leads to an increased GLUN2B localization in the TIF leaving the total amount of GLUN2B unaltered while doesn’t alter GLUN2A localization in TIF (Student’s t-test, P = 0.0275 N = 4). ( C ) Confocal images of neurons live stained for GLUN2A, fixed and counterstained against β3-tubulin. Relative quantification of density of GLUN2A puncta are shown on the left (Mann-Whitney Rank Sum Test, P = 0.070) and mean intensity of GLUN2A clusters on the right (Mann-Whitney Rank Sum Test, P = 0.001). ( D ) Images of neurons live stained for GLUN2B and quantification of GLUN2B puncta density (Mann-Whitney Rank Sum Test, P = 0.003) and mean intensity (Student’s t-test, P = 0.610). ( E , F ) Images of neurons live stained for GLUN2A ( E ) or GLUN2B ( F ), fixed and counterstained against the presynaptic marker bassoon. Right histograms show quantification of GLUN2A positive synapses (Mann-Whitney Rank Sum Test, P ≤ 0.001) and GLUN2A puncta mean intensity (Mann-Whitney Rank Sum Test, P = 0.005) (number of analyzed puncta: control = 1010, FTY720 = 720; number of analyzed fields: control = 35; FTY720 = 30) or GLUN2B positive synapses (Mann-Whitney Rank Sum Test, P = 0.005) and GLUN2B puncta mean intensity (number of analyzed puncta: control = 1250, FTY720 = 807; number of analyzed fields: control = 37; FTY720 = 32). ( G ) Representative images of spines co-transfected with SEP-GluN2A(green) and dTom (red) acquired at the indicated time point in control and FTY720-treated neurons. ( H ) XY graph representing the DMFI/MFI 0 of SEP-GluN2A over time (number of analysed spines: control = 40, FTY720 = 47; Mann-Whitney Rank Sum Test, P = 0.037 at t 50 and P = 0.009 at t 60 ). ( I ) Images of spines co-transfected with SEP-GluN2B and dTom as in G. ( J ) XY graph representing the DMFI/MFI 0 of SEP-GluN2B over time (number of analysed spines: control = 15, FTY720 = 22; Mann-Whitney Rank Sum Test, P = 0.030 at t 60 ).
    Figure Legend Snippet: FTY720 modulates GLUN2B localization in the postsynaptic fraction and increases surface GLUN2B and GLUN2A expression at synapses. ( A , B ) Western blotting of the homogenate (HOMO), postsynaptic triton insoluble fraction (TIF) and triton soluble fraction (TSF) obtained from control and FTY720-treated hippocampal neurons (DIV14). Tubulin is used as loading control, while PSD-95 as a marker of postsynaptic fraction. FTY720 leads to an increased GLUN2B localization in the TIF leaving the total amount of GLUN2B unaltered while doesn’t alter GLUN2A localization in TIF (Student’s t-test, P = 0.0275 N = 4). ( C ) Confocal images of neurons live stained for GLUN2A, fixed and counterstained against β3-tubulin. Relative quantification of density of GLUN2A puncta are shown on the left (Mann-Whitney Rank Sum Test, P = 0.070) and mean intensity of GLUN2A clusters on the right (Mann-Whitney Rank Sum Test, P = 0.001). ( D ) Images of neurons live stained for GLUN2B and quantification of GLUN2B puncta density (Mann-Whitney Rank Sum Test, P = 0.003) and mean intensity (Student’s t-test, P = 0.610). ( E , F ) Images of neurons live stained for GLUN2A ( E ) or GLUN2B ( F ), fixed and counterstained against the presynaptic marker bassoon. Right histograms show quantification of GLUN2A positive synapses (Mann-Whitney Rank Sum Test, P ≤ 0.001) and GLUN2A puncta mean intensity (Mann-Whitney Rank Sum Test, P = 0.005) (number of analyzed puncta: control = 1010, FTY720 = 720; number of analyzed fields: control = 35; FTY720 = 30) or GLUN2B positive synapses (Mann-Whitney Rank Sum Test, P = 0.005) and GLUN2B puncta mean intensity (number of analyzed puncta: control = 1250, FTY720 = 807; number of analyzed fields: control = 37; FTY720 = 32). ( G ) Representative images of spines co-transfected with SEP-GluN2A(green) and dTom (red) acquired at the indicated time point in control and FTY720-treated neurons. ( H ) XY graph representing the DMFI/MFI 0 of SEP-GluN2A over time (number of analysed spines: control = 40, FTY720 = 47; Mann-Whitney Rank Sum Test, P = 0.037 at t 50 and P = 0.009 at t 60 ). ( I ) Images of spines co-transfected with SEP-GluN2B and dTom as in G. ( J ) XY graph representing the DMFI/MFI 0 of SEP-GluN2B over time (number of analysed spines: control = 15, FTY720 = 22; Mann-Whitney Rank Sum Test, P = 0.030 at t 60 ).

    Techniques Used: Expressing, Western Blot, Marker, Staining, MANN-WHITNEY, Transfection

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 93
    Alomone Labs anti nr2b
    Leptin signaling increases pNR2B Y1472 levels and surface expression. ( A ) Representative Western blot of hippocampal neurons treated with leptin (50 nM), PP1 (10 µM), or both for 2 hours. ( B ) Quantification of pNR2B Y1472 intensity normalized to total <t>NR2B</t> intensity (n = 3). ( C ) Representative Western blot of hippocampal protein extracts from P10 wild-type and ob/ob mice pups (wild-type: n = 5; ob/ob : n = 5). ( D ) Quantification of pNR2B Y1472 intensity normalized to total NR2B intensity and total NR2B intensity normalized to the neuronal marker MAP2B intensity (n = 3). ( E ) Representative Western blot of surface biotinylated hippocampal cultures treated with leptin (50 nM, 2 hours). Biotinylated proteins were affinity purified (AP) with streptavidin magnetic beads. ( F ) Quantification of biotinylated NR2B intensity normalized to NR2B intensity in total lysate (n = 3). All experiments were repeated in 3 independent culture preparations and expressed as the mean ± SEM, * P
    Anti Nr2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti nr2b/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti nr2b - by Bioz Stars, 2022-07
    93/100 stars
      Buy from Supplier

    95
    Alomone Labs rabbit rb antibodies against glun2b
    ECM digestion increases <t>p1472-GluN2B</t> level and decreases the endocytosis of GluN2B. ( A )Dissociated hippocampal cultures at DIV21-24 were treated with Hya over night and endocytosed GluN2B (green) was quantified using Map2 staining as mask (red). ( B ) There is less endocytosis of GluN2B after ECM removal within 30 minutes (Ctl 1.00 ± 0.02, n = 79; Hya 0.9 ± 0.02, n = 80; average ± SEM, Unpaired t-test, **P = 0.0015. Scale bar: 5 µm). ( C ) Quantitative WB from lysates of acute hippocampal slices treated with Ctl or Hya probed with an antibody against pGluN2B pTyr1472 (AP2 binding site) and GluN2B. ( D ) Quantification of WB of acute hippocampal slices and cortical cultures (DIV 21–24) revealed that the amount of phosphorylated GluN2B, normalized to the total amount of GluN2B, is increased after Hya treatment (overnight for cultures, 3 h for slices; slices: Ctl 1.00 ± 0.06, n = 4; Hya 1.23 ± 0.09, n = 4; cultures: Ctl 1.00 ± 0.05, n = 9; Hya 1.26 ± 0.1, n = 9; Unpaired t-test, cultures: P = 0.0332, slices P = 0.0837, ***P
    Rabbit Rb Antibodies Against Glun2b, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit rb antibodies against glun2b/product/Alomone Labs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rabbit rb antibodies against glun2b - by Bioz Stars, 2022-07
    95/100 stars
      Buy from Supplier

    Image Search Results


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

    Journal: Endocrinology

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

    doi: 10.1210/endocr/bqz030

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

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

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

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

    Journal: Endocrinology

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

    doi: 10.1210/endocr/bqz030

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

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

    Techniques: Expressing, Western Blot, Transfection

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

    Journal: Endocrinology

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

    doi: 10.1210/endocr/bqz030

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

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

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

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

    Journal: Endocrinology

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

    doi: 10.1210/endocr/bqz030

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

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

    Techniques: Expressing, Transfection

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-07003-3

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-07003-3

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

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

    Techniques: CTL Assay, Blocking Assay

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-017-07003-3

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

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

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

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-69454-5

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

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

    Techniques: Marker

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

    Journal: Scientific Reports

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

    doi: 10.1038/s41598-020-69454-5

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

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

    Techniques: Diffusion-based Assay

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

    Journal: Frontiers in Aging Neuroscience

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

    doi: 10.3389/fnagi.2021.782737

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

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

    Techniques: Mouse Assay, Immunofluorescence

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

    Journal: Frontiers in Aging Neuroscience

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

    doi: 10.3389/fnagi.2021.782737

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

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

    Techniques: Immunohistochemistry, Mouse Assay