Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old) incubated with BDNF (50 ng/mL). Synaptoneurosomes were immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1, PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( A ) were analyzed for the GluN2B integrated density ( B ) and GluN2B mean gray value ( C ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, in at least three independent experiments performed in different preparations. ****p < 0.0001, by Kruskal-Wallis’s test and Dunn’s multiple comparisons test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) that were stimulated with BDNF (50 ng/ml for 10 or 30 min), as indicated. Neurons were then live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A) , were analyzed for the total number ( B ), intensity ( C ), and area ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per density of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the means of the control and are the means ± SEM of 43-45 cells per condition, from at least three independent experiments performed in different preparations. *p < 0.05, **p < 0.01 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Control, Analysis, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Representative images of hippocampal neurons (DIV 14 - 15) pre-incubated with GÖ 6983 (100 nM) or vehicle (DMSO; 1:1000 dilution for 40 min) and then either maintained under the same conditions or stimulated with BDNF (50 ng/ml for 30 min), as indicated. Neurons were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus, fixed, and then further immunoassayed for PSD-95, vGlut1, and MAP2. Scale bar, 5 μm. Images illustrated in ( A ) were analyzed for the total number ( B ), area ( C ), and intensity ( D ) of surface GluN2B puncta per µm. Synaptic (PSD-95- and vGlut1-colocalized) surface GluN2B number ( E ), area ( F ), and intensity ( G ) of puncta per µm of excitatory synapses (number of puncta PSD-95–vGlut1 colocalized per µm), were also analyzed. Data are normalized to the mean of the DMSO control and are the means ± SEM of 28 - 30 cells per condition, in at least three independent experiments performed in different preparations. ***p < 0.001, ****p < 0.0001 by one-way analysis of variance (ANOVA) followed by Bonferroni post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Incubation, Control, Analysis, Immunofluorescence

Journal: bioRxiv

Article Title: Synaptic accumulation of GluN2B-containing NMDA receptors mediates the effects of BDNF-TrkB signalling on synaptic plasticity and in epileptogenesis

doi: 10.1101/2024.10.21.618702

Figure Lengend Snippet: ( A ) Experimental design for the lithium-pilocarpine model of Status Epilepticus. ( B ) Representative images of hippocampal synaptoneurosomes (prepared from adult Sprague-Dawley rats 6-8 weeks old, treated with saline, saline and ANA-12, Pilocarpine or Pilocarpine and ANA-12). Synaptoneurosomes were live-immunoassayed for GluN2B using an antibody against an extracellular epitope in the GluN2B N-terminus and immunoassayed for vGlut1 and PSD-95. Merge scale bar, 10 μm. Insert scale bar, 0.5 µm. Images illustrated in ( B ) were analyzed for the GluN2B integrated density ( C ) and GluN2B mean gray value ( D ). Data are the means ± SEM of 779 - 840 synaptoneurosomes per condition, from at least four animals for each experimental condition. ****p < 0.0001, **p<0.01 as determined by Kruskal Wallis’s test and Dunn’s multiple comparisons test. Representative western blot and analysis ( E-G ). Proteins were extracted from synaptoneurosomes prepared from the same animals used in the immunocytochemistry experiments. For the immunoblot, antibodies against pTrkB, total TrKB and β-Tubulin were used. In this analysis, pTrkB levels were normalized to total TrkB levels and total TrkB levels were normalized to β-tubulin. Data are means ± SEM of at least four animals for each experimental condition. *p < 0.05, by one-way analysis of variance (ANOVA) followed by Dunnett’s post-test.

Article Snippet: To label surface GluN2B-containing NMDAR, live neurons (low-density hippocampal cultures) were incubated for 10 min at room temperature (RT) with an antibody against an extracellular epitope of the GluN2B N-terminus (1:100; AGC-003, Alomone Labs) diluted in a saline buffer (145 mM NaCl, 5 mM glucose, 10 mM HEPES, 5 mM KCl, 1.8 mM CaCl 2 , 1 mM MgCl 2, [pH 7.3]) (Sham) solution, as previously described ( ).

Techniques: Saline, Western Blot, Analysis, Immunocytochemistry, Immunofluorescence

Journal: Channels

Article Title: Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats

doi: 10.1080/19336950.2020.1871205

Figure Lengend Snippet: List of antibodies used for immunohistochemistry

Article Snippet: Rabbit anti-GluN2D , 1:1000 , Alomone Labs , ACG-020.

Techniques: Immunohistochemistry

Journal: Annals of neurology

Article Title: Absence of Neuronal Autoantibodies in Neuropsychiatric Systemic Lupus Erythematosus.

doi: 10.1002/ana.25908

Figure Lengend Snippet: Figure 1. Clinical features and immunoglobulin G (IgG) binding to conformational neuronal surface epitopes from plasma and cerebrospinal fluid (CSF) of patients with neuropsychiatric lupus (NPSLE). A. Clinical features of 35 patients with SLE, including 15 with active NPSLE at the time of sampling. 32/34 (94%) were on immunotherapies, including prednisolone (71%), hydroxychloroquine (41%), belimumab (6%) and rituximab (3%). ANA = anti-nuclear antibody. B. Live cell-based assays employing HEK293T cells with surface expressed N-methyl D-aspartate receptors (NMDARs) as NR1-NR2A or NR1-NR2B heteromers (nuclei highlighted with DAPI, 4′,6-diamidino-2-phenylindole). First panel shows that a commercial antibody directed against the extracellular domain of the NR2A-subunit (red) binds to HEK293T cells which express NR1-NR2A heteromers. NMDAR-antibody encephalitis (NMDAR-Ab-E) patient serum IgGs (n=11) bind to the NR1-NR2A heteromers (second panel), and to NR1-NR2B heteromers / NR1 homomers (data not shown). NMDAR-antibody patient CSFs show similar binding (as Irani et al, 2010).10 Serum from healthy controls (n=36), plasma and CSFs from SLE patients (n=35 of each) show no binding. No binding was observed with the G11 antibody (data not shown, lower panel representative). Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone,

Article Snippet: Throughout, IgG binding was visualized with an anti-human 568-Alexaflour antibody (1:750 dilution, A-21090) C. Commercial antibodies against the extracellular domains of NR1 (Alomone, AGC-001), NR2A (Alomone, AGC-002) and NR2B (kind gift from Prof Stephenson, London) subunits bound to the surface of live HEK293T cells co-transfected with EGFP plus NR1 and NR2B subunits.

Techniques: Binding Assay, Clinical Proteomics, Sampling

Journal: The EMBO Journal

Article Title:

doi: 10.1038/s44318-025-00484-3

Figure Lengend Snippet: ( A ) Overview of instrumental conditioning of DDHD2 −/− and C57BL/6 (Wild Type) mice. ( B ) PCA plot of hippocampal proteins displaying significantly altered expression in response to instrumental conditioning in either DDHD2 −/− or wild-type mice. ( C ) Quadrant plot comparing the significant learning responsive hippocampal protein expression in DDHD2 −/− and wild-type mice. Proteins coloured light red display increased expression upon ablation of DDHD2, while dark red coloured proteins have decreased expression. ( D ) Segmented sunburst plot displaying the synaptic GO cellular component terms of the above-mentioned responsive proteins (learning-induced proteins dysregulated by DDHD2 genetic ablation). ( E ) Bubble plot of significantly up- and down-regulated pathways when comparing instrumentally conditioned DDHD2 −/ − and wild-type hippocampal proteomes using reactome geneset analysis (RGSA). The average log fold change of associated proteins is displayed on the x - axis. The number of proteins detected in the pathway is depicted by bubble size and position on the y -xis. ( F ) Network plot displaying the change in GluN1 NMDA receptor subunit interactome between DDHD2 −/− and wild-type instrumentally conditioned mice. Box colour indicates Log fold change expression, with red denoting increased expression and blue decreased expression. ( G ) Western blotting analysis of hippocampal lysates probed for GluN1. Bar plot of GluN1 band densitometry normalised to β-actin. Data information: Experiments were performed using four biological replicates. ( B ) The criteria for identifying proteins with significantly altered expression and PCA conditions were as described for Fig. . ( C ) Protein expression dysregulation was determined by subtracting the instrumental responsive change in DDHD2 −/− animals by the expression change in wild-type controls. Proteins displaying values >3 or <0.33 were considered dysregulated in the hippocampus of DDHD2 −/− animals. ( E ) Pathway enrichment analysis was performed using the RGSA PADOG algorithm. Pathways with p values < 0.05 were considered significantly enriched. ( F ) Hippocampal proteins were considered differentially expressed between the DDHD2 −/− and wild-type instrumentally conditioned animals if the p value for the t -test comparison intensity values was ≤0.05 and log fold change was ≥0.25. GluN1 interactors among the differentially expressed proteins was determined using the STRING protein interaction database with a combined interaction score cut off ≥0.4. ( G ) Western blotting experiments for the GluN1 NMDA receptor subunit were performed on hippocampal lysates obtained from three separate animals per treatment condition. Error bars indicate the SEM. Statistical analysis was performed using two-way ANOVA comparing (i) instrumental conditioning ( p = 0.101), (ii) DDHD2 status ( p = 0.007) and (iii) interaction effect ( p = 0.026). Pairwise comparison instrumental condition effect group means was performed using Fisher’s least significant difference test: C57BL/6 wild type p = 0.012; DDHD2 −/− p = 0.561.

Article Snippet: GluN1 , Alomone Labs , AGC-001-GP.

Techniques: Expressing, Western Blot, Comparison

Journal: The EMBO Journal

Article Title:

doi: 10.1038/s44318-025-00484-3

Figure Lengend Snippet: ( A ) Schematic displaying the proposed pathway impacting protein enrichment in membranes based on pharmacological inhibition of DDHD2 (KLH-45) and NMT1/2 (IMP-1088). ( B ) Proteomics analysis of isolated cortical neuron membrane fractions immediately after cLTP in the presence of NMT1/2 inhibitor IMP-1088 and the DDHD2 inhibitor KLH-45. ( C ) Western blot analysis for subcellular markers in the low- and high-density membrane fractions generated using ultracentrifugation. ( D – F ) Volcano plots showing cLTP-induced protein enrichment and depletion compared to APV-treated controls in vehicle (dark grey), IMP-1088 (green) and KLH-45 (red) treated cortical neurons, respectively. Positive values indicate enrichment in the high-density membrane fraction whereas negative value indicate a depletion. The y- axis indicates the −log 10 Student t -test p values, with values exceeding 1.3 considered significant. ( G ) Principal component analysis plot of proteins displaying altered membrane localisation as determined by one-way ANOVA. ( H ) Quadrant plot comparing the effect of IMP-1088 and KLH-45 treatment on proteins displaying disrupted enrichment in high-density membrane fractions compared to the controls (cLTP log 2 fold change observed in the inhibitor – cLTP log 2 fold change observed in vehicle). Correlation analysis identified proteins with similar inhibitor-induced changes upon IMP-1088 and KLH-45 treatment (purple data points). Proteins with poor correlations between inhibitor effects are represented in grey. ( I ) Line plot displaying the relative protein expression changes of highly correlated inhibitor-effected proteins in cLTP-induced enrichment in high-density membrane fractions compared to the vehicle treated neurons. Negative values depict a decreased membrane enrichment compared to the cLTP effect in vehicle controls, whereas positive values indicate an increased enrichment. ( J ) GluN1 synaptic surface expression analysis using a GluN1 extracellular Ab (magenta) and a PSD95 intracellular Ab (red) in neurons 10 min post-cLTP or APV treatment. Bar plot: KLH-45 (red) and IMP-1088 (green) treatments reduce GluN1 surface expression following cLTP. ( K ) Bar plot displaying the relative intensity of the NMDA receptor GluN1 subunit detected in the high-density membrane fraction from each treatment condition using untargeted proteomics. Data Information: Experiments were performed using three biological replicates. ( D – F ) Statistical analysis of cLTP-induced changes in high-density membrane fraction enrichment was performed using pairwise analysis of cLTP/APV signal intensities for each treatment condition (vehicle, IMP-1088 and KLH-45 treated) by Student t -test. P values ≤ 0.05 (≥1.3 −log 10 ) and log 2 fold change ≥0.25 were considered significant. ( G ) Principal component analysis was performed on proteins displaying differential membrane partitioning as determined by one-way ANOVA, where p values of ≤0.05 were considered significant. PCA compared the highest 2 Eigenvalues >1 as the two plotted dimensions. ( H ) One-way ANOVA significant proteins ( p ≤ 0.05) displaying correlated inhibitor-induced disruption to enrichment in high-density membrane fractions was determined as follows: The effect of each inhibitor was determined by subtracting the log 2 relative change in protein intensity in cLTP vehicle from relative cLTP-induced change observed in the inhibitor-treated neurons (log 2 [cLTP veh/APV veh] - log 2 [cLTP inhibitor/APV veh]). Proteins displaying an inhibitor effect ≥0.25 were considered to have altered enrichment in the high-density membrane fraction. Commonalities between inhibitor treatments were determined by dividing the IMP-1088 effect by the KLH-45 effect and selecting proteins displaying a ratio between 0.3 and 3. Correlations were statistically confirmed using the Pearson correlation test, with R coefficients greater than 0.7 considered highly correlated. ( J ) Analysis was performed over two replicates of cLTP treatments with ten regions of interest (ROIs) analysed per condition. Error bars indicate the SEM. Statistical significance was determined using an ordinary one-way ANOVA with post hoc pairwise analysis using Šídák’s multiple comparisons test between APV and cLTP for each group. cLTP-induced change p values: Vehicle p = 0.998; KLH-45 treated p = 0.002; IMP-1088 treated p = 0.0006. Asterisks indicate multiple comparisons between APV and cLTP treatment, ** p < 0.01, *** p < 0.001. ( K ) Error bars indicate the SEM. Statistical analysis was performed using two-way ANOVA comparing (i) cLTP effect ( p = 0.0007), (ii) inhibitor treatment ( p = 0.6543) and (iii) interaction effect ( p = 0.012). Pairwise comparison of the cLTP of each treatment group was performed using Fisher’s least significant difference test: Vehicle p = 0.331; KLH-45 p = 0.229; IMP-1088 p = 0.0001. *** p ≤ 0.0001.

Article Snippet: GluN1 , Alomone Labs , AGC-001-GP.

Techniques: Protein Enrichment, Inhibition, Isolation, Membrane, Western Blot, Generated, Expressing, Disruption, Comparison

Journal: International Journal of Molecular Sciences

Article Title: NR1 and NR3B Composed Intranuclear N -methyl- d -aspartate Receptor Complexes in Human Melanoma Cells

doi: 10.3390/ijms19071929

Figure Lengend Snippet: Tables of antibodies used in the experiments.

Article Snippet: Anti-NR3B , rabbit, polyclonal , 1:250 , 1:50 , Alomone Labs, Jerusalem, Israel AGC-031.

Techniques: