anti nr2b  (Alomone Labs)

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

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

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

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

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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 (

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

Techniques: Labeling, Diffusion-based Assay

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

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

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

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

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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.

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

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

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

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

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

Techniques: Fluorescence, Imaging, Staining

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

Techniques: Diffusion-based Assay, Fluorescence

Journal: The EMBO Journal

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

doi: 10.1002/embj.201386356

Figure Lengend 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

Article Snippet: As previously described (Groc et al , , , ; Bats et al , ; Heine et al , ), quantum dots (QD) 655 goat F(ab')2 anti-rabbit IgG (Invitrogen) were first incubated for 30 min with 1 μl of the polyclonal antibodies against GluN2A (Alomone Labs; epitope corresponding to residues 41–53 of GluN2A subunit) or GluN2B subunits (Alomone Labs; epitope corresponding to residues 323–337 of GluN2B subunit).

Techniques: Fluorescence

Journal: bioRxiv

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

doi: 10.1101/2021.09.30.462507

Figure Lengend Snippet: TG2 localises extracellularly in primary astrocytes and at synaptic sites in neurons. 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

Article Snippet: Immunocytochemical stainingCells 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

Journal: Gastroenterology

Article Title: N-methyl D-Aspartate Channels Link Ammonia and Epithelial Cell Death Mechanisms in Helicobacter pylori Infection

doi: 10.1053/j.gastro.2011.08.048

Figure Lengend Snippet: NMDA channel subunit NR2B expression in surface, parietal, and chief cells is transcriptionally regulated in HP-infected tissues. Paraffin-embedded tissues from ( A ) sham- (Contr-20 wkPI) or ( B ) 6 and ( C ) 20 wkPI HP-infected mice were stained for the NR2B

Article Snippet: Filters were incubated with anti-NR2B (Alomone Labs, Jerusalem, Israel), anti-BAX (BD Pharmingen, San Jose, CA), or anti-BAK (Santa Cruz, Santa Cruz, CA).

Techniques: Expressing, Infection, Mouse Assay, Staining