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: 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: Translational Psychiatry

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

doi: 10.1038/s41398-021-01549-7

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

Article Snippet: Schematically, neurons were incubated with either rabbit polyclonal anti-GluN2B (Alomone Laboratories; 1:200) or mouse monoclonal anti-GFP (1:500, Roche, Switzerland), followed by an incubation with QD655 coupled to goat anti-rabbit F(ab’)2 (1:10000, Invitrogen).

Techniques: Diffusion-based Assay, Inhibition

Journal: Translational Psychiatry

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

doi: 10.1038/s41398-021-01549-7

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

Article Snippet: Schematically, neurons were incubated with either rabbit polyclonal anti-GluN2B (Alomone Laboratories; 1:200) or mouse monoclonal anti-GFP (1:500, Roche, Switzerland), followed by an incubation with QD655 coupled to goat anti-rabbit F(ab’)2 (1:10000, Invitrogen).

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

Journal: Translational Psychiatry

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

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: NMDAR surface dynamics and synaptic plasticity are developmentally impaired in the MAM model. a Schematic description of the experimental workflow. Cultured hippocampal networks were made from rat pups. Examples of immuncytochemical staining of neurons (NeuN, green), nucleus (DAPI, blue), and glial cells (GFAP, red) in cultured network at 10 days in vitro from control or MAM-exposed pups. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons from control or MAM-exposed pups at three developmental stages: days in vitro 6–10 (D8), days in vitro 10–15 (D12), days in vitro 15–22 (D20). Scale bars = 5 µm/300 nm (left/right). Right, Comparison of GluN2B-NMDAR diffusion coefficient control or MAM-exposed conditions (D8 cont, n = 1916 trajectories; D8 MAM, n = 1220; D12 cont, n = 2055; D12 MAM, n = 946; D20 cont, n = 1288; D20 MAM, n = 1317; ** p

Article Snippet: Schematically, neurons were incubated with either rabbit polyclonal anti-GluN2B (Alomone Laboratories; 1:200) or mouse monoclonal anti-GFP (1:500, Roche, Switzerland), followed by an incubation with QD655 coupled to goat anti-rabbit F(ab’)2 (1:10000, Invitrogen).

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

Journal: Translational Psychiatry

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

doi: 10.1038/s41398-021-01549-7

Figure Lengend Snippet: DISC1 downregulation alters NMDAR surface dynamics. a Schematic description of the experimental workflow: we compared the GluN2B-NMDAR surface dynamics onto neurons in which we downregulated either IL1RAPL1 or DISC1. b Representative trajectories (50 ms acquisition) of surface GluN2B-NMDAR-QD complexes on neurons transfected with either a scrambled siRNA (DISC1 Scr) or DISC1 siRNA (DISC1 Kd). Scale bar = 300 nm. Below , Cumulative distributions of synaptic GluN2B-NMDAR instantaneous diffusion coefficient (µm 2 /s) in DISC1 Scr and DISC1 Kd (Scr, n = 976 trajectories; Kd, n = 662; *** p

Article Snippet: Schematically, neurons were incubated with either rabbit polyclonal anti-GluN2B (Alomone Laboratories; 1:200) or mouse monoclonal anti-GFP (1:500, Roche, Switzerland), followed by an incubation with QD655 coupled to goat anti-rabbit F(ab’)2 (1:10000, Invitrogen).

Techniques: Transfection, Diffusion-based Assay

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