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

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

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

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, Immunocytochemistry

Journal: Frontiers in Molecular Neuroscience

Article Title: A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology

doi: 10.3389/fnmol.2022.994513

Figure Lengend Snippet: E1577K impairs surface expression of NR2B . (A) Representative regions of secondary dendrites with GFP-cell fill and NR2B surface stain. Insets show a single spine from each condition. Scale bar -10 μm. (B) Number of NR2B surface puncta normalized to total dendrite (GFP) area measured. (C) Average surface NR2B puncta size. (D) Average intensity of NR2B surface puncta. N = 2, 5 cells per replicate. (E) Regions of dendrites stained with Kalirin-7 and total NR2B to assess colocalization following overexpression of indicated constructs. Deconvoluted insets indicate Kalirin-7 and NR2B puncta adjacent within indicated spines. * p < 0.05. ** p < 0.01. *** p < 0.005.

Article Snippet: Proteins were separated by SDS PAGE, immobilized on PVDF by Western blot, and visualized [Flag M2 (Sigma #F1804), NR2B (Novus Biologicals, #NB100-74475), Kalirin-spectrin (Sigma #02122)].

Techniques: Expressing, Staining, Over Expression, Construct

Journal: Frontiers in Molecular Neuroscience

Article Title: A developmental delay linked missense mutation in Kalirin-7 disrupts protein function and neuronal morphology

doi: 10.3389/fnmol.2022.994513

Figure Lengend Snippet: E1577K shows impaired Rac1-GEF activity. (A) Western Blot of anti-FLAG pulldown of Kalirin-7 and E1577K from HEK293T, with co-immunoprecipitation of transfected NR2B (Mock = non-transfected). (B) HEK293T cells were transfected with the indicated conditions and active-Rac1 was pulled down from lysates with PAK-PBD coated Sepharose beads. Positive (+ ve) and Negative (-ve) samples indicate Mock transfected lysates Loaded with GTP-γ-S and GDP, respectively. Blots were probed with anti-FLAG and anti-Rac1. (C) Quantification of Active-RAC1 pulldown under the indicated conditions. * p < 0.05.

Article Snippet: Proteins were separated by SDS PAGE, immobilized on PVDF by Western blot, and visualized [Flag M2 (Sigma #F1804), NR2B (Novus Biologicals, #NB100-74475), Kalirin-spectrin (Sigma #02122)].

Techniques: Activity Assay, Western Blot, Immunoprecipitation, Transfection

Journal: Cancers

Article Title: NMDA Receptor Signaling Mediates cFos Expression via Top2β-Induced DSBs in Glioblastoma Cells

doi: 10.3390/cancers11030306

Figure Lengend Snippet: NMDAR signaling impacts cFos expression in primary glioblastoma multiforme (GBM) cells. ( a ) Immunofluorescence staining of the GluN1 and GluN2B subunits of the NMDAR in the G1702 cells. Notably, GluN2B subunits are localized at the end of cellular protrusions (GluN1/GluN2B = green, Hoechst 33342 = blue; scale bar: 25 µm). ( b ) Immunofluorescence staining of 53BP1 (red) and Top2β (green) in G1702 cells. Top2β and 53BP1 form foci which partly co-localize (scale bar: 20 µm). ( c ) Relative cFos/GAPDH expression in G1702 cells treated with 1 mM Glu and 20 µM MK801, 20 µM ifenprodil or 1 µM ICRF193 overnight, analyzed through western blotting (error bars show SD, n = 3, one sample t -test, p > 0.05 (ns), p ≤ 0.05 (*), p ≤ 0.01 (**), p ≤ 0.001 (***)).

Article Snippet: The following primary antibodies were used: Rabbit anti-GluN1 (1:100, D65B7 Cell Signaling), mouse anti-GluN2B (1:200, S59-20 Stress Marq), rabbit anti-53BP1 (1:1000; H-300 Santa Cruz) and mouse anti-Top2β (1:100; A-12 Santa Cruz).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Additive Effects of Environmental Enrichment and Ketamine on Neuropathic Pain Relief by Reducing Glutamatergic Activation in Spinal Cord Injury in Rats

doi: 10.3389/fnins.2021.635187

Figure Lengend Snippet: Primary antibodies for western blotting.

Article Snippet: Rabbit anti-phospho-NMDAR2B Tyr 1472 (p-NR2B) , 1:1,000 , Merck Millipore, Darmstadt, Germany.

Techniques: Western Blot

Journal: Neural Regeneration Research

Article Title: Protective effect of tetrahydroxy stilbene glucoside on learning and memory by regulating synaptic plasticity

doi: 10.4103/1673-5374.191223

Figure Lengend Snippet: Effect of TSG on protein expression of Fyn and phosphorylation of NR2B in CA1 region of the hippocampus of AD model mice. Fyn and p-NR2B mediated amyloid-beta-induced synaptic plasticity damage in parallel. Amyloid-beta can inhibit Fyn kinase-mediated phosphorylation pathway of NR2B, while TSG can improve learning and memory abilities of the transgenic rats through increasing NR2B receptors and Fyn expression. Data are expressed as the mean ± SD ( n = 20). All data were compared by one-way analysis of variance followed by post-hoc Scheffe's test. * P < 0.05, vs . normal control group; # P < 0.05, vs . AD group. TSG: Tetrahydroxy stilbene glucoside; AD: Alzheimer's disease.

Article Snippet: The membrane was blocked with 5% skim milk, and incubated with primary antibodies, including rat anti-mouse Fyn monoclonal antibody (1:1,000; Stressgen, Victoria, Canada), rat anti-NR2B polyclonal antibody (1:1,000; Stressgen), rat anti-phospho-NR2B polyclonal antibody (1:1,000, Thermo Fisher Scientific Inc., St. Louis, MO, USA) and β-actin (1:1,000; Abcam, Cambridge, UK).

Techniques: Expressing, Phospho-proteomics, Transgenic Assay, Control

Journal: PLoS ONE

Article Title: Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

doi: 10.1371/journal.pone.0045575

Figure Lengend Snippet: RT-PCR images for NR2B mRNA extracted from the hippocampus of every group at 1 (D1), 3 (D3), 5 (D5), or 7 (D7) day postoperation. NR2B mRNA values were first normalized with GAPDH of the same sample, and then expressed as mean relative values compared with the sham group (set to 1). ** P<0.01 vs sham group at the same time point; # P<0.05, ## P<0.01 vs HIR group at the same time point.

Article Snippet: Following deparaffinization, the 4 μm-thick paraffin sections were boiled in 0.1 mol/L sodium citrate buffer (pH 6.0) for 20 min, using primary antibodies against NR2B (1∶500, Abbiotec, American) for 30 min at 37°C.

Techniques: Reverse Transcription Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

doi: 10.1371/journal.pone.0045575

Figure Lengend Snippet: A,Werstern blot images of NR2B protein extracted from the hippocampus of every group at 1 (D1), 3 (D3), 5 (D5), or 7 (D7) day afte operation. B, Statistical analysis of the relative optical density normalized to housekeeping gene β-actin (mean±SD,N = 3). * P<0.05, ** P<0.01 vs sham group at the same time point; # P<0.05, ## P<0.01 vs HIR group at the same time point; ★ P<0.01 vs HIR+60sen group at the same time point.

Article Snippet: Following deparaffinization, the 4 μm-thick paraffin sections were boiled in 0.1 mol/L sodium citrate buffer (pH 6.0) for 20 min, using primary antibodies against NR2B (1∶500, Abbiotec, American) for 30 min at 37°C.

Techniques:

Journal: PLoS ONE

Article Title: Senegenin Attenuates Hepatic Ischemia-Reperfusion Induced Cognitive Dysfunction by Increasing Hippocampal NR2B Expression in Rats

doi: 10.1371/journal.pone.0045575

Figure Lengend Snippet: Representative photomicrographs of the CA1 area of hippocampus illustrating expression of NR2B from sham group (A) at day 1 postoperation, or HIR group tested at day 1 (B), or 7 (C) postoperation or from HIR+15 (D), HIR+30 (E), or HIR+60 (F) groups tested at day 7 postoperation.

Article Snippet: Following deparaffinization, the 4 μm-thick paraffin sections were boiled in 0.1 mol/L sodium citrate buffer (pH 6.0) for 20 min, using primary antibodies against NR2B (1∶500, Abbiotec, American) for 30 min at 37°C.

Techniques: Expressing