Journal: bioRxiv

Article Title: Epithelial N-methyl-D-aspartate (NMDA) receptors mediate renal vasodilation by affecting kidney autoregulation

doi: 10.1101/2023.12.04.569973

Figure Lengend Snippet: NMDAR RNA expression along the nephron. The mandatory Grin 1 subunit is expressed in all the renal segments, especially in the medulla regions of the nephron. In the medulla region all NMDAR subunit are expressed, while in the cortex in addition to Grin1, Grin2D is the majority expressed in the distal nephron in addition to grin2C and Grin3A in DCT and in less magnitude in CNT and CCD.TMP: Transcript per millon. [PTS1] initial segment of proximal tubule; [PTS2] straight segment of proximal tubule; [PTS3] last segment of the proximal straight tubule in the outer stripe of outer medulla; [DTL1] short descending limb of the loop of Henle; [DTL2] long descending limb of the loop of Henle in the outer medulla; [DTL3] long descending limb of the loop of Henle in the inner medulla; [ATL]) thin ascending limb of the loop of Henle; [MTAL] medullary thick ascending limb of the loop of Henle; [CTAL] cortical thick ascending limb of the loop of Henle; [DCT] distal convoluted tubule; [CNT] connecting tubule, cortical collecting duct [CCD], outer medullary collecting duct [OMCD], and inner medullary collecting duct [IMCD])

Article Snippet: Tissue incubation with antibodies against GluN1 (StressMarq, S308-48) at 1:5000 dilution was performed overnight at 4°C.

Techniques: RNA Expression

Journal: Translational Psychiatry

Article Title: Synergistic inhibition of histone modifiers produces therapeutic effects in adult Shank3 -deficient mice

doi: 10.1038/s41398-021-01233-w

Figure Lengend Snippet: A , B Bar graphs showing mRNA levels of NMDAR subunits ( Grin1, Grin2a, Grin2b ) and AMPAR ( Gria1, Gria2 ) subunits ( A ) and synaptic proteins ( Shank3 , Homber1b , Arc ) ( B ) in PFC of adult saline-treated WT or Shank3 +/ΔC mice treated with saline or Rom/GSK (0.25/5 mg/kg, i.p., 3x) at 8 days post-treatment. n = 7–11 mice per group. C, D Bar graphs showing total ( C ) or synaptic ( D ) protein levels of NMDAR subunits in the 3 groups at 8 days post-treatment. C, n = 7–9 mice/group; D, n = 5–7 mice per group. Insets: representative immunoblots. In all figures, # p < 0.1, * p < 0.05, **p < 0.01, *** p < 0.001.

Article Snippet: Western blots were performed using antibodies against NR1 (1:500, NeuroMab, 75‐ 272), NR2A (1:500, Millipore, 07‐632), NR2B (1:500, Millipore, 06‐600), PSD95 (1:1000, Cell Signaling, 36233 S) and tubulin (1:5000 Sigma, T9026).

Techniques: Saline, Western Blot

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: Neural Regeneration Research

Article Title: Mechanisms responsible for the effect of median nerve electrical stimulation on traumatic brain injury-induced coma: orexin-A-mediated N-methyl-D-aspartate receptor subunit NR1 upregulation

doi: 10.4103/1673-5374.184494

Figure Lengend Snippet: Effect of MNS on NR1 expression in the prefrontal cortex of rats with TBI-induced coma (western blot assay). Control group: Sham-operated rats (skin incision but no TBI). TBI group: Weight-drop method was used to establish a model of TBI. Stimulated group: Comatose rats with TBI underwent MNS. Antagonist group: Comatose rats with TBI and given MNS received an intracerebroventricular injection of the OX1R antagonist SB334867. Data are expressed as the mean ± SD ( n = 6 rats per time point per group). * P < 0.05, vs . control group; # P < 0.05, vs . TBI group; † P < 0.05, vs . stimulated group; § P < 0.05, vs . 6 h; ‡ P < 0.05, vs . 12 h (one-way analysis of variance). MNS: Electrical stimulation of the median nerve; TBI: traumatic brain injury; h: hours.

Article Snippet: The membranes were incubated with rabbit anti-NR1 polyclonal antibody (1:200; Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei Province, China) and mouse anti-β-actin monoclonal antibody (1:500; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4°C, and washed in Tris buffered saline with Tween 20.

Techniques: Expressing, Western Blot, Control, Injection

Journal: Neural Regeneration Research

Article Title: Mechanisms responsible for the effect of median nerve electrical stimulation on traumatic brain injury-induced coma: orexin-A-mediated N-methyl-D-aspartate receptor subunit NR1 upregulation

doi: 10.4103/1673-5374.184494

Figure Lengend Snippet: Effect of MNS on NR1 immunoreactivity in the prefrontal cortex of rats with TBI-induced coma (× 400). Control group: Sham-operated rats (skin incision but no TBI). TBI group: Weight-drop method was used to establish a model of TBI. Stimulated group: Comatose rats with TBI underwent MNS. Antagonist group: Comatose rats with TBI and given MNS received an intracerebroventricular injection of the OX1R antagonist SB334867. Brown cells (red arrows) are NR1-immunoreactive. Positive immunostaining for NR1 was found in the cytoplasm, cell membrane, and nucleus of neurons in the prefrontal cortex. MNS: Electrical stimulation of the median nerve; TBI: traumatic brain injury; h: hours.

Article Snippet: The membranes were incubated with rabbit anti-NR1 polyclonal antibody (1:200; Wuhan Boster Biotechnology Co., Ltd., Wuhan, Hubei Province, China) and mouse anti-β-actin monoclonal antibody (1:500; Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China) overnight at 4°C, and washed in Tris buffered saline with Tween 20.

Techniques: Control, Injection, Immunostaining, Membrane

Journal: Scientific Reports

Article Title: Furin inhibitor protects against neuronal cell death induced by activated NMDA receptors

doi: 10.1038/s41598-018-23567-0

Figure Lengend Snippet: ( a ) Cortical cells were fixed at 10 DIV and stained for NeuN (green) and GFAP (red) and with Hoechst (blue). The scale bar represents 100 µm. ( b ) The number of NeuN- and GFAP-positive cells was counted. Results were expressed as the percentage of these cells among the total number of Hoechst-positive cells and as the means ± SE in 4 independent experiments. ( c ) Proteins from cultured cortical neurons at 2, 4, 6, 8, 10 and 12 DIV were analyzed by immunoblotting with anti-GluN1, anti-GluN2A, anti-GluN2B, and anti-β-actin antibodies. Bands corresponding to GluN1, GluN2A, and GluN2B were scanned, the scanned bands were normalized by each protein at 2 DIV on the same blot. β-actin was used as a loading control. Results are the means ± SE (n = 4 independent experiments).

Article Snippet: The following primary antibodies were used: mouse anti-β-actin (a5441, Sigma), mouse anti-GluN1 (556308, BD Biosciences, Franklin Lakes, NJ, USA), rabbit anti-GluN2A (AB1555P, Millipore), mouse anti-GluN2B (610416, BD Biosciences), and rabbit anti-calpain-2 (39165, Abcam).

Techniques: Staining, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Ethanol place preferences is associated with the expression of mRNA from GluN2A but not from GluN1 subunit in the NAc. A , Schematic of the experimental design. B , Place preference elicited by the administration of 70 nmol of ethanol in the pVTA. Data are mean ± SEM represented as preference score (test minus pretest time spent in ethanol-paired compartment). * denotes significant differences between means (p<0.01, t-Test). C , Diagram of brain coronal sections indicating in blue the area of all microinjections in pVTA. D , Expression of mRNA from NMDA subunits GluN1 and GluN2A in NAc after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell. * denotes significant differences between means (p<0.05). E , Expression of mRNA from NMDA subunits GluN1 and GluN2A in hippocampus after ethanol induced CPP. Data are mean ± SEM represented as number of dots (mRNA molecules) per cell.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Expressing

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative pictures of the compilation files obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc (left) and hippocampus (right) after ethanol induced CPP. Squares delimit the area of the amplified pictures shown in for the NAc and in for the hippocampus. Abbreviatures: Aca , anterior commissure; CA3 , field CA3 of hippocampus; DG , dentate gyrus; CA1 , field CA1 of hippocampus.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: In Situ Hybridization, Amplification

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in NAc. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: bioRxiv

Article Title: Dose-Dependent Induction Of CPP Or CPA By Intra-pVTA Ethanol: Role Of Mu Opioid Receptors And Effects On NMDA Receptors

doi: 10.1101/2020.01.07.897702

Figure Lengend Snippet: Representative amplified pictures obtained in the mRNA In Situ Hybridization Assay used for the quantification of mRNA from GluN1 and GluN2A subunits in hippocampus. Amplified pictures from one animal of each group (aCSF on the left and ethanol 70 nmol on the right) were selected.

Article Snippet: Membranes were then blocked in 5% non-fat dried milk in TBS-Tween-20 (TBS-T) 0.1% (20mM Tris and 500mM NaCl pH 7.5) and incubated overnight at 4°C with the primary antibody: anti-GluN1 (1:1000; Merck KGaA, Darmstadt, Germany) and anti-GluN2A monoclonal antibody (1:1000; Merck KGaA, Darmstadt, Germany).

Techniques: Amplification, In Situ Hybridization

Journal: International Journal of Molecular Medicine

Article Title: Direct binding of Toll-like receptor 4 to ionotropic glutamate receptor N-methyl-D-aspartate subunit 1 induced by lipopolysaccharide in microglial cells N9 and EOC 20

doi: 10.3892/ijmm.2017.3331

Figure Lengend Snippet: Lipopolysaccharide (LPS) triggers interaction of Toll-like receptor 4 (TLR4) and glutamate receptor N-methyl-D-aspartate subunit 1 (GluN1) in N9 and EOC 20 microglial cells. N9 (A and E) and EOC 20 (B and F) microglial cells were treated with LPS (1 µ g/ml) for various durations. Cell surface expression of TLR4 was blotted with anti-TLR4 (A and B, top panel). Total cell lysates were blotted with anti-GluN1 (E and F, top panel) or anti-β-actin (A, B, E and F, bottom panel). β-actin blots exhibited equal loading. Scanning densitometry values of western blotting for TLR4 (C and D) and GluN1 (G and H) were normalized by densitometry of corresponding β-actin bands and expressed as mean ± standard error of the mean. The mean was calculated from four independent experiments (n=4). * P<0.05 vs. prior to LPS treatment. Co-immunoprecipitation of TLR4 and GluN1 in N9 (I) and EOC 20 (J) microglial cells treated with LPS (1 µ g/ml) for various durations. Anti-TLR4 immunoprecipitates were blotted with anti-GluN1. Similar results were obtained from three independent experiments (n=3).

Article Snippet: The rabbit anti-GluN1 antibody (cat. no. LS-B13901) used in immunochemistry was purchased from LifeSpan BioSciences (Seattle, WA, USA).

Techniques: Expressing, Western Blot, Immunoprecipitation

Journal: International Journal of Molecular Medicine

Article Title: Direct binding of Toll-like receptor 4 to ionotropic glutamate receptor N-methyl-D-aspartate subunit 1 induced by lipopolysaccharide in microglial cells N9 and EOC 20

doi: 10.3892/ijmm.2017.3331

Figure Lengend Snippet: Toll-like receptor 4 (TLR4) and glutamate receptor N-methyl-D-aspartate subunit 1 (GluN1) co-localize in N9 and EOC 20 microglial cells. (A–L) N9 and (M–X) EOC 20 microglial cells were treated with 1 µ g/ml LPS for (D and P) 30 min, (E and Q) 1 h, (F and R) 2 h or (G and S) 4 h, and stained for GluN1 (green) and TLR4 (red). Without LPS treatment, (C and O) no co-localization of TLR4 and GluN1 was observed. Single GluN1 and TLR4 images are shown in (A and M) and (B and N), respectively, in the group without LPS treatment. No signal was observed if TLR4 and GluN1 antibodies were replaced with goat IgG and rabbit IgG, respectively. Five to ten fields from each condition were randomly selected and analyzed with CoLocalizer Pro software and Protein Proximity Analysis software. The results are shown in (I–L) for N9 and (U–X) for EOC 20 microglial cells. * P<0.05 vs. 0 (without LPS treatment). Scale bar, 20 µ M. The experiments were repeated at least three times with similar results, and two coverslips were examined for each condition in one test (n=6). Col., colocalization; Rr, Pearson's correlation coefficient; LPS, lipopolysaccharide.

Article Snippet: The rabbit anti-GluN1 antibody (cat. no. LS-B13901) used in immunochemistry was purchased from LifeSpan BioSciences (Seattle, WA, USA).

Techniques: Staining, Software

Journal: International Journal of Molecular Medicine

Article Title: Direct binding of Toll-like receptor 4 to ionotropic glutamate receptor N-methyl-D-aspartate subunit 1 induced by lipopolysaccharide in microglial cells N9 and EOC 20

doi: 10.3892/ijmm.2017.3331

Figure Lengend Snippet: The binding of Toll-like receptor 4 (TLR4) and glutamate receptor N-methyl-D-aspartate subunit 1 (GluN1) was investigated with antibody-based fluorescence resonance energy transfer (FRET) in N9 and EOC 20 microglial cells. (A–F) N9 and (G–L) EOC 20 microglial cells were treated with 1 µ g̸ml lipopolysaccharide (LPS) for 30 min and stained for TLR4 (red) and GluN1 (green). (A and G) Pre-bleach and (B and H) post-bleach images for TLR4. (C and I) Pre-bleach and (D and J) post-bleach images for GluN1. (F and L) FRET efficiency images. When one cell was randomly selected as the region of interest 1 (ROI1), four other cells and background were selected as controls, which were not bleached. (E and K) Five cells as ROI1 were analyzed from each coverslip and FRET efficiency was calculated. * P<0.05 vs. ROI1. Similar results were obtained from at least three independent experiments and two coverslips were examined for each condition in one test (n=6). Scale bar, 20 µ M.

Article Snippet: The rabbit anti-GluN1 antibody (cat. no. LS-B13901) used in immunochemistry was purchased from LifeSpan BioSciences (Seattle, WA, USA).

Techniques: Binding Assay, Fluorescence, Förster Resonance Energy Transfer, Staining

Journal: International Journal of Molecular Medicine

Article Title: Direct binding of Toll-like receptor 4 to ionotropic glutamate receptor N-methyl-D-aspartate subunit 1 induced by lipopolysaccharide in microglial cells N9 and EOC 20

doi: 10.3892/ijmm.2017.3331

Figure Lengend Snippet: Metabotropic glutamate receptor 5 (mGluR5) antagonist attenuates the binding of Toll-like receptor 4 (TLR4) and glutamate receptor N-methyl-D-aspartate subunit 1 (GluN1) induced by lipopolysaccharide (LPS) in N9 and EOC 20 microglial cells. (A and B) N9 and (C and D) EOC 20 microglial cells were pretreated with 0.1 mM 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine (MTEP) for 30 min, and then treated with 1 µ g/ml LPS for 1 h. Anti-TLR4 immunoprecipitates from (A) N9 or (C) EOC 20 microglial cells were blotted with anti-GluN1. Total cell lysates from (B) N9 or (D) EOC 20 microglial cells were blotted with anti-TLR4 (B and D, top panel), anti-GluN1 (B and D, middle panel), or anti-β-actin (B and D, bottom panel). Similar results were obtained from four independent experiments (n=4).

Article Snippet: The rabbit anti-GluN1 antibody (cat. no. LS-B13901) used in immunochemistry was purchased from LifeSpan BioSciences (Seattle, WA, USA).

Techniques: Binding Assay