Journal: Ecotoxicology and environmental safety

Article Title: Influence of bifenthrin exposure at different gestational stages on the neural development.

doi: 10.1016/j.ecoenv.2023.115365

Figure Lengend Snippet: Fig. 5. Effect of BF treatment at GD 16-B on the protein levels of VGluT1, VGAT, and NMDA receptors in mouse hippocampi at 6-week-old. (A) Repre sentative western blot images of VGluT1, VGAT, NR1, NR2A and NR2B expression in the mouse hippocampi at 6-week-old. These western blot images are from one membrane. Taking β-Tubulin as loading control. (B) & (C) Quantization of the protein levels of VGluT1, VGAT, VGluT1/VGAT, NR1, NR2A and NR2B of the male mouse (B) and female mouse hippocampi (C) at 6-week-old. N = 4 (litter). The bands in each column of Fig. 5A are from the same pup. Unpaired Student’s t-test was used to analyze the statistical significance. * * P < 0.01 vs. respective sex control.

Article Snippet: Primary antibodies against VGluT1 (#CL2754), VGAT (#131003), NMDA Receptor 2B (NR2B, GluN2B, #21920–1-AP) were bought from Novus Biologicals (Littleton, CO, USA), synaptic systems (Gottingen, Germany), and Proteintech (Chicago, IL, USA) respectively.

Techniques: Western Blot, Expressing, Membrane, Control

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: Differential surface expression of GluN2B C-terminal variants expressed in cultured rat hippocampal neurons. A, Three rare variants in the GluN2B C terminus were identified from human patients via third-generation deep sequencing. Human S1415 is analogous to S1413 in rat; L1424 is analogous to L1422 in rat; and S1452 is analogous to S1450 in rat. B, Reference and minor allele frequency of three C-terminal variants in human diseases. C, D, Three rat GluN2B mutants are expressed after transient transfection in DIV 14 hippocampal neurons. Surface expression of GluN2B S1413L is reduced, whereas other GluN2B variants do not alter surface expression in hippocampal neurons. C, Representative images of surface (red channel) and intracellular (blue channel) expression of rat GluN2B mutants. Scale bar, 10 μm. D, Quantitive analysis of C. **p < 0.01, n = 5, one-way ANOVA.

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques: Expressing, Cell Culture, Sequencing, Transfection

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: Binding to MAGUKs is impaired in three rat GluN2B mutants. The GST-GluN2B C terminus fusion protein was used for a pull-down assay of PSD-95 (A, C) and SAP102 (B, D) from mouse brain synaptosomal fractions. *p < 0.05; **p < 0.01, p < 0.001, n = 5, one-way ANOVA.

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques: Binding Assay, Pull Down Assay

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: Dendritic spine number is decreased in hippocampal pyramidal neurons expressing GluN2B S1413L. A, DIV 14 hippocampal neurons were transiently transfected with pCAG-GFP and GluN2B. At DIV 17, neurons were stained for GFP-tagged GluN2B and spines filled with GFP in 20 μm dendrites were analyzed. Three regions were selected and averaged for every neuron. Scale bar, 5 μm. B, Quantification of spine density. ***p < 0.001, n = 3, one-way ANOVA.

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques: Expressing, Transfection, Staining

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: The GluN2B C-teminal variants do not change functional properties of NMDARs. Current responses in A–D were recorded under TEVC from Xenopus oocytes to determine pharmacological properties. A, B, Concentration–response curves (VHOLD = −40 mV) for glutamate in the presence of 30 μm glycine (A) and glycine in the presence of 100 μm glutamate (B) are shown. C, Concentration–response curves for inhibition of mutant and wild-type NMDA receptors by extracellular Mg2+; currents (VHOLD = −60 mV) were activated by 100 μm glutamate and 30 μm glycine. D, Mean ratio (in percentage) of the current response to 100 μm glutamate and 30 μm glycine recorded at pH 6.8 and the current response at pH 7.6 (VHOLD = −40 mV). E, Superimposed whole-cell current recordings in response to 1000 μm glutamate and 30 μm glycine from HEK cells expressing GluN1/GluN2B (VHOLD = −60 mV). F, Weighted mean time constants describing deactivation were not detectably different among the mutations tested. p > 0.05, one-way ANOVA, Bonferroni's multiple-comparisons test.

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques: Functional Assay, Concentration Assay, Inhibition, Mutagenesis, Expressing

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: Summary of pharmacological results of GluN2B WT and mutants in X. laevis oocytes

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques:

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: The three GluN2B C-terminal mutants do not change the response properties of NMDARs

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques:

Journal: The Journal of Neuroscience

Article Title: A Rare Variant Identified Within the GluN2B C-Terminus in a Patient with Autism Affects NMDA Receptor Surface Expression and Spine Density

doi: 10.1523/JNEUROSCI.0827-16.2017

Figure Lengend Snippet: GluN2B S1413L cannot rescue NMDA EPSCs. A, Loss of evoked NMDA EPSCs in Cre-positive GRIN2Afl/flGRIN2Bfl/fl hippocampal CA1 neurons at P13–P15. Amplitude: Control (Cnt), 71.9 ± 9.9 pA; Cre, 4.2 ± 0.9 pA, n = 10. ***p < 0.0001. B, Coexpression of Cre-mCherry with GluN2B-IRES-GFP rescued NMDA EPSCs Amplitude: Cnt, 71.8 ± 12.2 pA; Cre+GluN2B, 71.9 ± 8.8 pA, n = 16. p = 0.99. C, Coexpression of Cre-mCherry with GluN2B S1413L–IRES-GFP did not fully rescue NMDA EPSCs [103 ± 8.5 pA; Cre + GluN2B(S1413L), 66 ± 7.9 pA]. n = 16. **p = 0.0042. D, GluN2B S1413L variant does not change the NMDA EPSC decay time constant compared with GluN2B. Averaged and peak-aligned NMDA EPSCs were recorded from control or transfected cells from GRIN2Afl/flGRIN2Bfl/fl mice [decay time constant: Cnt, 220.0 ± 9.4 ms, n = 32; Cre + GluN2B, 265.9 ± 16.4 ms, n = 16; Cre + GluN2B(S1413L), 268.2 ± 11.3, n = 16. Cnt vs Cre + GluN2B, p = 0.012 < 0.05; Cnt vs Cre + GluN2B(S1413L), p = 0.0033 < 0.01; Cre + GluN2B vs Cre + GluN2B(S1413L), p = 0.91]. Scale bar, 50 pA and 0.02 s. Error bars indicate SEM.

Article Snippet: Protein expression was induced by IPTG; the bacteria were collected, lysed, and GST-GluN2B fusion proteins present in the supernatant were enriched by isolating the fusion proteins on glutathione Sepharose-4B beads (GE Healthcare).

Techniques: Variant Assay, Transfection

Journal: Cell Death & Disease

Article Title: Alpha-synuclein is involved in manganese-induced spatial memory and synaptic plasticity impairments via TrkB/Akt/Fyn-mediated phosphorylation of NMDA receptors

doi: 10.1038/s41419-020-03051-2

Figure Lengend Snippet: a , b After treatment with Mn, western blotting was used to measure the level of α-Syn overexpression in the hippocampus. c – f The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) were measured by western blotting in vivo. n = 6. g , h HT22 cells were transfected with LV-α-Syn shRNA, and the cytotoxicity was measured using the CCK-8 assay and lactate dehydrogenase (LDH) release assay. i – m The levels of phosphorylated NMDA receptors (GluN2B and GluN2A) and α-Syn expression were measured by western blotting in vitro. n = 4. ** P < 0.01 compared to their control counterparts; # # P < 0.01, and # P < 0.05 for comparison between the Mn treatment group.

Article Snippet: PSD95, SynGAP, β-actin, phospho-CaMKII (Thr286), CaMKII-α, NMDA receptor 2A (GluN2A), NMDA receptor 2B (GluN2B), Fyn, phospho-NMDA receptor 2A (GluN2A) (Tyr1246), and phospho-NMDA receptor 2B (GluN2B) (Tyr1472) primary antibodies were purchased from Cell Signaling Technology.

Techniques: Western Blot, Over Expression, In Vivo, Transfection, shRNA, CCK-8 Assay, Lactate Dehydrogenase Assay, Expressing, In Vitro, Control, Comparison

Journal: Cell Death & Disease

Article Title: Alpha-synuclein is involved in manganese-induced spatial memory and synaptic plasticity impairments via TrkB/Akt/Fyn-mediated phosphorylation of NMDA receptors

doi: 10.1038/s41419-020-03051-2

Figure Lengend Snippet: a – e After normal or transfected HT22 cells were pretreated with brain-derived neurotrophic factor (BDNF) and Mn, the levels of phospho-Akt and phospho-Fyn, and Akt and Fyn expression were evaluated by western blotting. f – j After normal or transfected HT22 cells were pretreated with BDNF and Mn, the levels of phospho-GluN2B and phospho-GluN2A, and the expression of GluN2B and GluN2A were evaluated by western blotting. n = 4. β-actin was used as a loading control. ** P < 0.01, and * P < 0.05 compared to their control counterparts; ## P < 0.01 compared to their Mn-treated cells; ΔΔ P < 0.01, and Δ P < 0.05 for comparison between their BDNF-pretreated and Mn-treated cells.

Article Snippet: PSD95, SynGAP, β-actin, phospho-CaMKII (Thr286), CaMKII-α, NMDA receptor 2A (GluN2A), NMDA receptor 2B (GluN2B), Fyn, phospho-NMDA receptor 2A (GluN2A) (Tyr1246), and phospho-NMDA receptor 2B (GluN2B) (Tyr1472) primary antibodies were purchased from Cell Signaling Technology.

Techniques: Transfection, Derivative Assay, Expressing, Western Blot, Control, Comparison

Journal: Cell Death & Disease

Article Title: Alpha-synuclein is involved in manganese-induced spatial memory and synaptic plasticity impairments via TrkB/Akt/Fyn-mediated phosphorylation of NMDA receptors

doi: 10.1038/s41419-020-03051-2

Figure Lengend Snippet: Mn-induced α-Syn protein overexpression contributes to further exacerbate brain-derived neurotrophic factor (BDNF) protein-level reduction and to inhibit TrkB/Akt/Fyn signaling, thereby disturbing Fyn-mediated phosphorylation of the NMDA receptor GluN2B subunit at tyrosine.

Article Snippet: PSD95, SynGAP, β-actin, phospho-CaMKII (Thr286), CaMKII-α, NMDA receptor 2A (GluN2A), NMDA receptor 2B (GluN2B), Fyn, phospho-NMDA receptor 2A (GluN2A) (Tyr1246), and phospho-NMDA receptor 2B (GluN2B) (Tyr1472) primary antibodies were purchased from Cell Signaling Technology.

Techniques: Over Expression, Derivative Assay, Phospho-proteomics

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate-induced NMDAR potentiation in HEK cells hinges on the expression of CaMKII and its interaction with the GluN2B subunit. (A) Representative isolated I NMDAR traces recorded from CaMKII-expressing HEK cells transiently transfected with expressing constructs encoding the NMDAR subunits GluN1 and Glu2B. Currents were evoked by puffs of glutamate/glycine at holding voltages ranging from +60 mV to -20 mM (in 20 mV decrement steps), recorded at 30-sec intervals during baseline (control), after 5-6 min in the presence of 10 mM lactate, during the washout period, or in the presence of 50 µM AP5. (B) Lactate stimulates peak I NMDAR in HEK cells expressing functional NMDAR (GluN1::GluN2B) in CaMKIIα-expressing HEK cells (right panels) but not in cells lacking CaMKIIα (WT, left panels). This effect is noticeable at holding potentials above 20 mV. In contrast, lactate prolongs decay times in both types of HEK cells, independent of CaMKIIα expression (bottom panels). (C) Summary bar charts of I NMDAR amplitudes (top) and decay times (bottom) ± SEM in response to puffs of co-agonists at a holding potential of +30 mV, in the presence of lactate or pyruvate in NMDAR-expressing HEK cells with or without CaMKIIα expression. (D) Time-course of peak I NMDAR in the absence and presence of 10 mM lactate for CaMKIIα-expressing HEK cells transfected with GluN1 and either WT or mutant GluN2B subunits. The two GluN2B variants (L1298/R1300Q and R1300Q/S1203D) are known to disrupt the interaction between GluN2B and CaMKIIα. (E, F) Quantitative summaries of the I NMDAR amplitudes (E) and decay times (F). Bar charts are expressed as percentages of averaged baseline values ± SEM. Statistical significance was determined as described in .

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Expressing, Isolation, Transfection, Construct, Control, Functional Assay, Mutagenesis

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate potentiation of NMDAR responses depends on two redox-sensitive cysteine-rich sequences in the intrinsically disordered CTD of GluN2B subunit (A, left panels) Representative traces of I NMDAR evoked by puffs of glutamate/glycine at the soma, recorded in cultured neurons at a holding potential of +30 mV. Traces during the baseline period are marked with a white circle, and those 5 and 8 minutes after the onset of the lactate treatment with a colored circle. Top example traces include 4 mM NADH in the intracellular patch pipette solution, while bottom traces do not. The representative traces with lactate were recorded 15 and 18 minutes after going whole-cell. (A, right panel) The time- course of I NMDAR amplitudes induced by co-agonists is shown before, during, and after lactate exposure, with (purple dots) and without (dashed line) intracellular NADH. The dashed line illustrates the time-course of I NMDAR amplitudes without NADH, as shown in . (B, left panels) Representative I NMDAR traces evoked by brief applications of NMDAR co-agonists were recorded in cultured neurons at a holding potential of +30 mV with a patch pipette solution containing the reductant DTT (top) or the oxidizer DTNB (bottom) during treatment with lactate (orange circle) or the baseline control period (white circle). (B, right panel) Bar chart summarizing the peak I NMDAR responses ± SEM in neurons exposed to lactate (10mM) with or without intracellular infusion of reductants (DTT, 1 mM and βME, 300 µM) or the DNTB (200 µM). (C) Prediction of intrinsic disorder by residue position in mouse GluN2B in reducing (plus, dark purple line) or oxidizing/normal (minus, red line) conditions. Redox- sensitive stretches are identified by the divergence of the red and dark purple lines across the disorder score threshold of 0.5. These regions are highlighted in light purple and marked by dashed rectangles that form areas highlighted in light purple delineated by dashed rectangles. The beige background indicates the C-terminal domain (CTD) of the protein. (D, top) Representative I NMDAR traces evoked by brief applications of NMDAR co-agonists, recorded at a holding potential of +30 mV in HEK cells in the presence of lactate (colored circles) or during the baseline control period (white circle). (D) Quantitative summaries of the effect of 10 mM lactate (expressed as percentage of baseline) on I NMDAR amplitudes in CaMKIIα- expressing HEK cells transfected with GluN1 and WT Glu2B (WT), C946-C954S mutant GluN2B (Δredox 1-CTD GluN2B), or C1215S-C1218S-C1239-C1242S-C1245S GluN2B (Δredox 2-CTD GluN2B) subunits. Summary data are expressed as percentages of averaged baseline values ± SEM. Statistical significance was determined as described in .

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Cell Culture, Transferring, Control, Residue, Expressing, Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Lactate potentiates the binding between CaMKII and GluN2B and drives a CaMKII-dependent increase of GluN2B-PSD-95 clustering. (A) Lactate increases the association between GluN2B and CaMKII. Representative western blots showing expression of CaMKII and GluN2B in total protein extract (input), CaMKII, GluN2B, p1303-GluN2B, and PSD-95 in synaptosome extract (synaptosomes), and CaMKII and GluN2B in synaptosome extracts immunoprecipitated with GluN2B antibody (IP: GluN2B) treated or not with 10 mM of lactate (Lac) or pyruvate (Pyr). (B) Quantitative summary plot for the synaptosome-enriched proteins immunoprecipitated with a GluN2B antibody expressed as a ratio of their respective control (Ctl) ± SEM. Statistical significance: * p < 0.05, Two-Way ANOVA followed by Dunnett’s multiple comparisons test. (C) Representative images of proximity ligation assays (PLA) for GluN2B/PSD-95 (green) in cultured cortical neurons treated with pyruvate (Pyr, 10 mM) or lactate (Lac, 10 mM) in the presence or absence of the CaMKII inhibitor peptide myr-AIP-2 (1 µM) or its scrambled control (scr, 1 µM). MAP2 immunostaining (blue) was used to delineate the dendrites. Scale bar, 5 µM. (D) Quantitative summary with data expressed as the mean ± SD. PLA cluster numbers per 10 µM of dendrite. ***p<0.001, One-Way ANOVA followed by Bonferroni’s multiple comparison tests.

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Binding Assay, Western Blot, Expressing, Immunoprecipitation, Control, Ligation, Cell Culture, Immunostaining, Comparison

Journal: bioRxiv

Article Title: Lactate potentiates NMDA receptor currents via an intracellular redox mechanism targeting cysteines in the C-terminal domain of GluN2B subunits: implications for synaptic plasticity

doi: 10.1101/2024.11.21.624499

Figure Lengend Snippet: Schematic representation of the cellular mechanisms involved in the modulation of NMDAR responses and facilitation of synaptic plasticity by lactate. (A) Extracellular lactate is transported passively along its concentration gradient through the monocarboxylate transporters 2 (MCT-2) into neurons (1) where it is converted by the lactate dehydrogenase (LDH) in presence of the oxidized form of nicotinamide adenine dinucleotide (NAD + ) to pyruvate and the reduced form of nicotinamide adenine dinucleotide (NADH) (2). This metabolic process increases the NADH/NAD+ ratio, leading to a more reduced intracellular milieu. Such a change in the internal redox state, in turn, affects cysteine-rich redox-sensitive sites on the C-terminal domain of the NMDAR GluN2B subunits favoring CaMKII binding (3) and modulates the activity of ryanodine receptors (RyRs), causing, together with the activation of NMDAR, an increase in intracellular Ca 2+ levels (4). Elevated intracellular Ca 2+ levels activate CaMKII which allow its binding to NMDAR (5). Pyruvate is imported into the mitochondria and metabolized by the tricyclic acid cycle and the oxidative phosphorylation to produce ATP to sustain the energy needs of neurons (6). (B) A detailed view of the molecular interaction between NMDARs and CaMKII under the influence of lactate. Lactate-induced NADH production alters the intracellular redox environment, promoting the rearrangement of the intrinsically disordered structure of redox-sensitive cysteine-rich sites within the C-terminal domain of GluN2B. These modifications, along with the activation of CaMKII by Ca 2+ , promote the binding of CaMKII to NMDARs, stimulating the accumulation of NMDAR at synapses, illustrated by an increase in the association between GluN2B and PSD-95, ultimately enhancing synaptic NMDAR function and synaptic plasticity (C).

Article Snippet: Cells were incubated overnight at 4°C with mouse anti-PSD-95 (1:1000, Abcam ab2723) and rabbit anti-GluN2B (1:1000, Cell Signaling Technology 14544) antibodies in 5% BSA in PBS.

Techniques: Concentration Assay, Binding Assay, Activity Assay, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Early and Delayed Impact of Nanosilver on the Glutamatergic NMDA Receptor Complex in Immature Rat Brain

doi: 10.3390/ijms22063067

Figure Lengend Snippet: The expression of GluN1 (NR1) subunit of NMDA receptor in the brain of control and AgNPs/Ag citrate-exposed immature rats in PND 35. ( A ) Representative confocal images of the cerebral cortex; triple immunostaining of GluN1 (red) with anti-MAP antibody (green) and Hoechst to visualize cell nuclei (blue); scale bars indicate 20 µm. ( B ) The mean intensity of the fluorescence; data are means ± SD from 9–10 sections from three distinct brains in each group, * p < 0.05 and ** p < 0.01 vs. control, # p < 0.05 vs. AgNPs. ( C ) Relative expression of GluN1 protein measured against β-actin as an internal standard. Representative immunoblot and graph illustrating the mean results obtained using four distinct animals. ( D ) Expression of GluN1 mRNA determined by qPCR and normalized against actin. Data in ( C , D ) are means ± SD from four experiments, * p < 0.05, ** p < 0.01 vs. control, # p < 0.05 vs. AgNPs (one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Sections were stained with primary anti-NMDA receptor 1 antibody (1:50; Cell Signaling; Cat. No. 5704) or anti-NMDA receptor 2B antibody (1:50; Proteintech; Cat. No. 21920-1-AP) and co-stained with mouse primary anti-MAP2 (1:750; Sigma Aldrich; Cat. No. M4403).

Techniques: Expressing, Control, Triple Immunostaining, Fluorescence, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Early and Delayed Impact of Nanosilver on the Glutamatergic NMDA Receptor Complex in Immature Rat Brain

doi: 10.3390/ijms22063067

Figure Lengend Snippet: The expression of GluN2B (NR2) subunit of NMDA receptor in the brain of AgNPs/Ag citrate-exposed immature rats in PND 35. ( A ) Representative confocal images of cerebral cortex; triple immunostaining of GluN2B (red) with anti-MAP antibody (green) and Hoechst to visualize cell nuclei (blue); scale bars indicate 20 µm. ( B ) The mean intensity of the fluorescence; data are means ± SD from n = 9–10 sections from three distinct brains in each group, ** p < 0.01 vs. control. ( C ) Relative expression of GluN2B protein measured against β-actin as an internal standard. Representative immunoblot and graph illustrating the mean results obtained using four distinct animals; ** p < 0.01 vs. control ( D ) Expression of GluN2B mRNA determined by qPCR and normalized against actin. Data are represented as means ± SD from four experiments, ** p < 0.01 significantly different vs. control (one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Sections were stained with primary anti-NMDA receptor 1 antibody (1:50; Cell Signaling; Cat. No. 5704) or anti-NMDA receptor 2B antibody (1:50; Proteintech; Cat. No. 21920-1-AP) and co-stained with mouse primary anti-MAP2 (1:750; Sigma Aldrich; Cat. No. M4403).

Techniques: Expressing, Triple Immunostaining, Fluorescence, Control, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Early and Delayed Impact of Nanosilver on the Glutamatergic NMDA Receptor Complex in Immature Rat Brain

doi: 10.3390/ijms22063067

Figure Lengend Snippet: The expression of GluN1 (NR1) subunit of NMDA receptor in the brain of AgNPs/Ag citrate-exposed immature rats in PND 90. ( A ) Representative confocal images of cerebral cortex; triple immunostaining of GluN1 (red) with anti-MAP antibody (green) and Hoechst to visualize cell nuclei (blue); scale bars indicate 20 µm. ( B ) The mean intensity of the fluorescence; data are means ± SD from n = 9–10 sections from three distinct brains in each group; there are no significant differences between groups. ( C ) Relative expression of GluN1 protein measured against β-actin as an internal standard. Representative immunoblot and graph illustrating the mean results obtained using four distinct animals. ( D ) Expression of GluN1 mRNA determined by qPCR and normalized against actin. Data are represented as means ± SD from four experiments, there are no statistical differences between groups (one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Sections were stained with primary anti-NMDA receptor 1 antibody (1:50; Cell Signaling; Cat. No. 5704) or anti-NMDA receptor 2B antibody (1:50; Proteintech; Cat. No. 21920-1-AP) and co-stained with mouse primary anti-MAP2 (1:750; Sigma Aldrich; Cat. No. M4403).

Techniques: Expressing, Triple Immunostaining, Fluorescence, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Early and Delayed Impact of Nanosilver on the Glutamatergic NMDA Receptor Complex in Immature Rat Brain

doi: 10.3390/ijms22063067

Figure Lengend Snippet: The expression of GluN2B (NR2) subunit of NMDA receptor in the brain of AgNPs/Ag citrate-exposed immature rats in PND 90. ( A ) Representative confocal images of cerebral cortex; triple immunostaining of GluN1 (red) with anti-MAP antibody (green) and Hoechst to visualize cell nuclei (blue); scale bars indicate 20 µm. ( B ) The mean intensity of the fluorescence; data are means ± SD from n = 9–10 sections taken from three distinct brains in each group; there are no significant differences between groups. ( C ) Relative expression of GluN2B protein measured against β-actin as an internal standard. Representative immunoblot and graph illustrating the mean results obtained using four distinct animals. ( D ) Expression of GluN2B mRNA determined by qPCR and normalized against actin. Data are represented as means ± SD from four experiments, there are no significant differences between groups (one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Sections were stained with primary anti-NMDA receptor 1 antibody (1:50; Cell Signaling; Cat. No. 5704) or anti-NMDA receptor 2B antibody (1:50; Proteintech; Cat. No. 21920-1-AP) and co-stained with mouse primary anti-MAP2 (1:750; Sigma Aldrich; Cat. No. M4403).

Techniques: Expressing, Triple Immunostaining, Fluorescence, Western Blot, Comparison

Journal: International Journal of Molecular Sciences

Article Title: Early and Delayed Impact of Nanosilver on the Glutamatergic NMDA Receptor Complex in Immature Rat Brain

doi: 10.3390/ijms22063067

Figure Lengend Snippet: The specific binding of radioligands to the membranes isolated from the forebrains of control, AgNP- and Ag citrate-exposed rats. Binding of the agonist [ 3 H]glutamate early after exposure at PND 35 ( A ) and late at PND 90 ( C ). Binding of the selective blocker of NMDA receptors [ 3 H]MK-801 early at PND 35 ( B ) and late at PND 90 after exposure ( D ). Data are means ± SD from three independent experiments, ** p < 0.01 vs. control (one-way ANOVA followed by Tukey’s multiple comparison test).

Article Snippet: Sections were stained with primary anti-NMDA receptor 1 antibody (1:50; Cell Signaling; Cat. No. 5704) or anti-NMDA receptor 2B antibody (1:50; Proteintech; Cat. No. 21920-1-AP) and co-stained with mouse primary anti-MAP2 (1:750; Sigma Aldrich; Cat. No. M4403).

Techniques: Binding Assay, Isolation, Control, Comparison

Journal: eLife

Article Title: KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization

doi: 10.7554/eLife.72483

Figure Lengend Snippet: ( A ) mEPSCs recorded from WT and cKO CA1 pyramidal neurons. n = 21 neurons per group; ***p < 0.001, Student’s t -test; For the cumulative probability curves, n.s. p > 0.05, ****p < 0.0001, Kolmogorov-Smirnov test. ( B ) Paired-pulse stimuli evoked EPSPs with different interval. n = 9 neurons per group, p > 0.05. The data were analyzed in separate t -tests at each stimulus intensity. ( C ) AMPAR and NMDAR EPSCs in CA1 pyramidal neurons of WT and cKO mice. Sample sweeps illustrating NMDAR-EPSCs recorded at +40 mV and AMPAR-EPSCs recorded at −70 mV. Histograms show ratio of NMDAR-EPSCs amplitude / AMPAR-EPSCs amplitude (right). n = 10 neurons, *p < 0.05. Student’s t -test. ( D ) Sample traces and input-output curve of NMDA receptor-mediated EPSCs between WT and cKO mice (n = 6 neurons, p > 0.05). The data were analyzed in separate t -tests at each stimulus intensity. ( E ) Sample traces and input-output curve of AMPA receptor-mediated EPSCs between WT and cKO mice (n = 6 neurons, *p < 0.05). The data were analyzed in separate t -tests at each stimulus intensity. ( F ) Cell-surface biotinylation shows a significant increase in GluA1 and GluA2 subunit surface expression in cKO hippocampus compared with WT. Histograms show percent change of GluA1, GluA2, GluN1, GluN2A, GluN2B subunits in total and biotinylation fraction. Flotillin was the internal control. n = 7 experiment repeats, *p < 0.05, **p < 0.01, Student’s t -test. ( G ) Example fEPSP before (baseline) and after (t = 60 min) 1× HFS stimulation in WT and cKO CA1 slices. Time course of percentage changes of fEPSP slope in WT and cKO CA1 slices. WT, n = 9 slices from 7 mice; cKO, n = 7 slices from 6 mice. Histogram shows fEPSP slope of WT and cKO neurons (55–60 min). *p < 0.05, Student’s t -test. ( H ) Example fEPSP before (baseline) and after (t = 135 min) 4× HFS stimulation in WT and cKO CA1 slices. Time course of percentage changes of fEPSP slope in WT and cKO CA1 slices. WT, n = 4 slices from 4 mice; cKO, n = 5 slices from 4 mice. Histogram shows fEPSP slope of WT and cKO neurons (130–135 min). *p < 0.05, Student’s t -test. ( I ) Time course of percentage changes of fEPSP slope before (baseline) and after low frequency stimulus (LFS) in control and cKO CA1 slices. WT, n = 7 slices from 5 mice; cKO, n = 7 slices from 6 mice. Histogram shows fEPSP slope of WT and cKO neurons (55–60 min). p = 0.7642, Student’s t -test. Figure 5—source data 1. Values for mEPSCs of WT and cKO neurons. Values for PPR ratio; Values for input-output curve of AMPAR and NMDAR mediated EPSCs between WT and cKO neurons; Values for NMDAR and AMPAR subunit surface expression; Values for percentage changes of fEPSCs amplitudes in WT and cKO neurons. Figure 5—source data 2. Original files of blots with the relevant bands.

Article Snippet: Antibody , Anti- GluN2B, (rabbit polyclonal) , Cell Signaling Technology , Cat# 4207, RRID: AB_1264223 , WB (1:2000).

Techniques: Expressing, Control

Journal: eLife

Article Title: KIF2C regulates synaptic plasticity and cognition in mice through dynamic microtubule depolymerization

doi: 10.7554/eLife.72483

Figure Lengend Snippet:

Article Snippet: Antibody , Anti- GluN2B, (rabbit polyclonal) , Cell Signaling Technology , Cat# 4207, RRID: AB_1264223 , WB (1:2000).

Techniques: Virus, SYBR Green Assay, Staining, Bicinchoninic Acid Protein Assay, Software, Sequencing, Recombinant

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: The changes of interstitial glutamate concentration, astrocytic glutamate reuptake function, and the expression of GluN2B, GluA1 and glutamate transporters in hippocampus after SAH at 8 weeks. (A) The concentration of interstitial glutamate in the hippocampus of the mice was detected by HPLC-MS/MS. **, P<0.01, the SAH group vs. the sham group. N=8 mice per group. (B) Glutamate uptake assay on the hippocampal astrocytes sorted by immunomagnetic beads. **, P<0.01, the SAH group vs. the sham group. N=6 mice per group. (C,D) Western blot was performed to detect the expression of p-GluN2B/GluN2B, p-GluA1/GluA1, GLT-1, and GLAST protein in hippocampal tissue in the sham group and the SAH group at 1 week before and 8 weeks after SAH. **, P<0.01, vs. the indicated groups. N=5 animals in each group.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Concentration Assay, Expressing, Tandem Mass Spectroscopy, Western Blot

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: Effects of selective HDAC2 inhibitor and GLT-1 inhibitor on DCI in SAH mice. (A) The SAH mice were intraperitoneally administered with HDAC2 inhibitor (2 mg/kg) and GLT-1 inhibitor (WAY-213613, 1 mg/kg) two weeks after surgery once every other day. The expression of GLT-1, HDAC2, p-GluN2B and p-GluA1 in hippocampus was detected after SAH at 8 weeks. N=5 animals in each group. (B) Forced swimming test and (C) sugar water preference test were used to evaluate the depressive behavior of SAH mice treated with HDAC2 inhibitor (Santacruzamate A) and GLT-1 inhibitor (WAY-213613). **, P<0.01, vs. the sham group; #, P<0.05 and ##, P<0.01, vs. the indicated groups. (D) Morris water maze test was used to detect the escape latency for reflecting spatial learning memory, *, P<0.01, and **, P<0.01, vs. the indicated groups. (E) Reference memory was detected by recording the time in target quadrant of Morris water maze test. **, P<0.01, vs. the sham group; #, P<0.05, vs. the indicated groups. N=8 mice per group.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Expressing

Journal: Annals of Translational Medicine

Article Title: Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1

doi: 10.21037/atm-20-4330

Figure Lengend Snippet: Negative regulation of GLT-1 by astrocytes HDAC2 leads to dysfunction of glutamate reuptake in the synaptic cleft. In normal condition, the acetylation of histones in astrocytes facilitates the transcriptional regulation of GLT-1. Glutamate in the synaptic cleft is rapidly absorbed into astrocytes to maintain excitability of synapses. The increase of HDAC2 in astrocytes after SAH results in the deacetylation of histones and inhibits the transcription expression of GLT-1. The decrease of GLT-1 expression will lead to the impairment of glutamate reuptake in astrocytes and the long-term accumulation of glutamate in the synaptic space, resulting the dephosphorylation of ionized glutamate receptors GluN2B and GluA1 on the postsynaptic membrane. These eventually result in the long-term inhibition of synaptic excitability and DCI. DCI, delayed cognitive impairment.

Article Snippet: The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967).

Techniques: Expressing, De-Phosphorylation Assay, Inhibition

Journal: Neuroscience

Article Title: Early Life Sleep Deprivation: Role of Oxido-Inflammatory Processes

doi: 10.1016/j.neuroscience.2019.02.021

Figure Lengend Snippet: The source and dilution of the antibodies used in western blotting experiments. Dilution for secondary antibodies is 1:2000.

Article Snippet: Protein Primary antibody Company Primary antibody Cat # Primary antibody Dilution Primary antibody RRID # Secondary antibody TNF-α Cell Signaling 3707 1:1000 AB_2240625 Goat anti-rabbit IL-6 Thermo Fisher Scientific ARC0062 1:1000 AB_J501203 Goat anti-mouse P-P38 MAPK Abeam Ab47363 1:1000 AB_881848 Goat anti-rabbit P38 MAPK Abeam Ab31828 1:1000 AB_881839 Goat anti-mouse P-JNK1,2,3 Abeam {"type":"entrez-nucleotide","attrs":{"text":"Ab124956","term_id":"46091695"}} Ab124956 1:1000 AB_10973183 Goat anti-rabbit JNK1,2,3 Abeam {"type":"entrez-nucleotide","attrs":{"text":"Ab208035","term_id":"94362026"}} Ab208035 1:1000 Goat anti-rabbit BDNF Santa Cruz SC-546 1:500 AB_630940 Goat anti-rabbit P-ERK 1/2 Cell Signaling 9106S 1:1000 AB_331768 Goat anti-mouse ERK 1/2 Cell Signaling 9107S 1:1000 AB_10695739 Goat anti-rabbit MKP1 Thermo fisher Scientific PA5–17973 1:1000 AB_10986429 Rabbit Anti-goat PSD95 Thermo fisher Scientific MA 1–045 1:1000 AB_325399 Goat anti-mouse GluA1 Cell Signaling D4N9V 1:1000 AB_2732897 Goat anti-rabbit GluN2b Abeam Ab65783 1:1000 AB_1658870 Goat anti-rabbit P-CaM KII Santa Cruz SC-32289 1:1000 AB_626786 Goat anti-mouse CaM KII Santa Cruz SC-9035 1:1000 AB_634551 Goat anti-rabbit P-CREB Cell Signaling 06–519 1:250 AB_626786 Goat anti-rabbit CREB Santa Cruz SC-58 1:1000 AB_631314 Goat anti-rabbit β -actin Cell Signaling 3700S 1:1000 AB_2242334 Goat anti-mouse Open in a separate window The source and dilution of the antibodies used in western blotting experiments.

Techniques: Western Blot

Journal: Neuroscience

Article Title: Early Life Sleep Deprivation: Role of Oxido-Inflammatory Processes

doi: 10.1016/j.neuroscience.2019.02.021

Figure Lengend Snippet: Examination of the protein expression levels of synaptic density/plasticity markers in the PFC. Protein levels of the AMPA receptor subunit (GluA1) in the PFC were measured at PND33 and PND90. The protein levels were normalized with the loading control β-actin (A). Protein levels of the NMDA receptor subunit (GluN2b) were measured in the PFC at PND33 and PND90. The protein levels were normalized with the loading control β-actin (B). Protein levels of the postsynaptic density protein (PSD95) in PFC at PND33 and PND90. The protein levels were normalized with the loading control β-actin (C). Phosphorylation levels of the calcium/calmodulin-dependent kinase II (CaMKII) were measured in the PFC at PND33 and PND90. The protein phosphorylation levels were normalized with total CaMKII (D). Bars are means ± standard deviation, n= 6–7 rats/group. Data were analyzed using two-tailed unpaired t-test. (*) significantly different at p<0.05. PND: Postnatal day. Group designations: control (CON), sleep deprivation (SD). Top panels are representative western blotting images.

Article Snippet: Protein Primary antibody Company Primary antibody Cat # Primary antibody Dilution Primary antibody RRID # Secondary antibody TNF-α Cell Signaling 3707 1:1000 AB_2240625 Goat anti-rabbit IL-6 Thermo Fisher Scientific ARC0062 1:1000 AB_J501203 Goat anti-mouse P-P38 MAPK Abeam Ab47363 1:1000 AB_881848 Goat anti-rabbit P38 MAPK Abeam Ab31828 1:1000 AB_881839 Goat anti-mouse P-JNK1,2,3 Abeam {"type":"entrez-nucleotide","attrs":{"text":"Ab124956","term_id":"46091695"}} Ab124956 1:1000 AB_10973183 Goat anti-rabbit JNK1,2,3 Abeam {"type":"entrez-nucleotide","attrs":{"text":"Ab208035","term_id":"94362026"}} Ab208035 1:1000 Goat anti-rabbit BDNF Santa Cruz SC-546 1:500 AB_630940 Goat anti-rabbit P-ERK 1/2 Cell Signaling 9106S 1:1000 AB_331768 Goat anti-mouse ERK 1/2 Cell Signaling 9107S 1:1000 AB_10695739 Goat anti-rabbit MKP1 Thermo fisher Scientific PA5–17973 1:1000 AB_10986429 Rabbit Anti-goat PSD95 Thermo fisher Scientific MA 1–045 1:1000 AB_325399 Goat anti-mouse GluA1 Cell Signaling D4N9V 1:1000 AB_2732897 Goat anti-rabbit GluN2b Abeam Ab65783 1:1000 AB_1658870 Goat anti-rabbit P-CaM KII Santa Cruz SC-32289 1:1000 AB_626786 Goat anti-mouse CaM KII Santa Cruz SC-9035 1:1000 AB_634551 Goat anti-rabbit P-CREB Cell Signaling 06–519 1:250 AB_626786 Goat anti-rabbit CREB Santa Cruz SC-58 1:1000 AB_631314 Goat anti-rabbit β -actin Cell Signaling 3700S 1:1000 AB_2242334 Goat anti-mouse Open in a separate window The source and dilution of the antibodies used in western blotting experiments.

Techniques: Expressing, Standard Deviation, Two Tailed Test, Western Blot

Journal: Scientific Reports

Article Title: PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B

doi: 10.1038/s41598-022-21488-7

Figure Lengend Snippet: Cellular distribution of NR2B and PSD-95 in the anterior cingulate cortex. Double immunofluorescence staining shows colocalization of NR2B with NeuN and PSD-95, but not with GFAP or Iba1. Scale bar = 100 μm. NR2B: NMDA receptor subunit 2B; NeuN: neuron-specific nuclear protein; GFAP: glial fibrillary acidic protein; Iba1: ionized calcium-binding adaptor molecule-1; PSD-95: postsynaptic density protein-95.

Article Snippet: The sections were blocked with 10% sheep or donkey serum for 2 h at room temperature and incubated with the following primary antibodies for 48 h at 4 °C: goat-anti-Iba1 (1:200, Abcam), mouse-anti-GFAP (1:500, Cell Signaling Technology), mouse-anti-NeuN (1:2000, Abcam), rabbit-anti-NR2B (1:800, Cell Signaling Technology), and mouse-anti-PSD-95 (1:100, Abcam).

Techniques: Double Immunofluorescence Staining, Binding Assay

Journal: Scientific Reports

Article Title: PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B

doi: 10.1038/s41598-022-21488-7

Figure Lengend Snippet: CCI-induced behavioral hypersensitivity, NR2B/PSD-95 upregulation, and CREB activation in the ACC. ( A ) Schematic diagram of the experiment. ( B ) The anatomical boundaries used for ACC tissue harvest. In comparison with the sham group, the CCI group showed significantly lower thermal latency ( C ) and mechanical threshold ( D ) on the 7th day after surgery (*** P < 0.001, independent t- test, n = 6). Western blot showing that compared to that in the sham group, the expression of NR2B ( E ) and PSD-95 ( F ) in the ACC was bilaterally increased in the CCI group (*** P < 0.001, ** P < 0.01, vs. Sham, one-way ANOVA, n = 4); while the total CREB ( G ) did not change, the level of pCREB ( H ) was bilaterally increased in the CCI group (*** P < 0.001, vs. Sham, one-way ANOVA, n = 4). GAPDH was loading control and error bars represent standard error of the mean (SEM). Original images of blots are available in supplementary Fig. . ACC: anterior cingulate cortex; CCI: chronic constriction injury; Ipsi: Ipsilateral; Contr: Contralateral; NR2B: NMDA receptor subunit 2B; CREB: Cyclic AMP response element-binding protein; pCREB: phosphorylated CREB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The sections were blocked with 10% sheep or donkey serum for 2 h at room temperature and incubated with the following primary antibodies for 48 h at 4 °C: goat-anti-Iba1 (1:200, Abcam), mouse-anti-GFAP (1:500, Cell Signaling Technology), mouse-anti-NeuN (1:2000, Abcam), rabbit-anti-NR2B (1:800, Cell Signaling Technology), and mouse-anti-PSD-95 (1:100, Abcam).

Techniques: Activation Assay, Western Blot, Expressing, Binding Assay

Journal: Scientific Reports

Article Title: PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B

doi: 10.1038/s41598-022-21488-7

Figure Lengend Snippet: PSD-95 AS-ODN attenuated behavioral hypersensitivity, inhibited the PSD-95/NR2B over-expression and CREB activation induced by CCI. ( A ) Schematic diagram of the experiment. ( B , C ) The location of ACC catheterization and microinjection. ( D , E ) In comparison with the PEI, the thermal latency and mechanical threshold were significantly higher in the AS-ODN group (* P < 0.05, *** P < 0.001, vs. CCI + PEI, two-way ANOVA, n = 6). ( F ) Western blot showed that the expression of PSD-95 in the AS-ODN group was significantly lower than that in the PEI group (* P < 0.05, vs. CCI + PEI, independent t- test, n = 3). ( G ) The expression of NR2B in the AS-ODN group was significantly lower than that in the PEI group (** P < 0.01, vs. CCI + PEI, independent t- test, n = 3). ( H , I ) The expression level of CREB was not altered significantly. The phosphorylated CREB was significantly lower than that in the PEI control group (*** P < 0.001, vs. CCI + PEI, independent t- test, n = 3). GAPDH was loading control, and error bars represent standard error of the mean (SEM). Original images of blots are available in supplementary Fig. . ACC: anterior cingulate cortex; CCI: chronic constriction injury; PEI: polyethyleneimine; AS-ODN: antisense oligonucleotide; PSD-95: postsynaptic density protein-95; NR2B: NMDA receptor subunit 2B; CREB: cyclic AMP response element-binding protein; pCREB: phosphorylated CREB; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The sections were blocked with 10% sheep or donkey serum for 2 h at room temperature and incubated with the following primary antibodies for 48 h at 4 °C: goat-anti-Iba1 (1:200, Abcam), mouse-anti-GFAP (1:500, Cell Signaling Technology), mouse-anti-NeuN (1:2000, Abcam), rabbit-anti-NR2B (1:800, Cell Signaling Technology), and mouse-anti-PSD-95 (1:100, Abcam).

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Binding Assay

Journal: Scientific Reports

Article Title: PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B

doi: 10.1038/s41598-022-21488-7

Figure Lengend Snippet: A schematic presentation of PSD-95 in the anterior cingulate cortex contributes to neuropathic pain by interdependent activation with NR2B. The material of the schematic diagram is provided by Figdraw.

Article Snippet: The sections were blocked with 10% sheep or donkey serum for 2 h at room temperature and incubated with the following primary antibodies for 48 h at 4 °C: goat-anti-Iba1 (1:200, Abcam), mouse-anti-GFAP (1:500, Cell Signaling Technology), mouse-anti-NeuN (1:2000, Abcam), rabbit-anti-NR2B (1:800, Cell Signaling Technology), and mouse-anti-PSD-95 (1:100, Abcam).

Techniques: Activation Assay