anti phospho n methyl d aspartate receptors subunit 2b nr2b tyr1472  (Cell Signaling Technology Inc)

Journal: Cells

Article Title: Simplified Procedure for Isolation and Culture of Neuronal Cells from Brains of Sickle Cell Mice

doi: 10.3390/cells15110976

Figure Lengend Snippet: Schematic overview of the isolation, culture, and analysis of hippocampal and cortical neurons from postnatal day 1 control (HbAA) and sickle cell (HbSS) mice. The workflow includes brain dissection, enzymatic dissociation, neuronal plating and maintenance, and downstream morphological and immunocytochemical analyses.

Article Snippet: Cells were fixed as described above at 3, 7, and 14 days in vitro (DIVs) with 2% paraformaldehyde in phosphate-buffered saline(Sigma-Aldrich, #158127) for 10 min at room RT and permeabilized with ice-cold 0.1% Triton X-100 (Sigma-Aldrich, #T8787) for 2 min; they were then blocked for 30 min with 3% donkey serum in PBS and incubated overnight at 4 °C with combinations of primary antibodies ( ): goat anti-RBFOX3/NeuN (1:200, Novus Biological, Centennial, CO, USA, #NBP3-05554), chicken anti-NF200 (1:3000, Neuromics, Edina, MN, USA, #CH22104), guinea pig anti-postsynaptic density (PSD) 95 (1:500, Alomone Labs, Jerusalem, Israel, #APZ-009-GP), rabbit anti-GluN2B (1:500, Alomone Labs, #AGC-003), chicken anti-MAP2 (1:2000, Thermo Fisher, #PA1-1005), and rabbit anti-VGLUT1 (1:300, Cell Signaling Technology, Danvers, MA, USA, #47181).

Techniques: Isolation, Analysis, Control, Dissection, Cell Culture, Phase Contrast, Immunofluorescence, Confocal Microscopy

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Expression of GluN2A (A) and GluN2B (B) and basal extracellular levels of L‐glutamate (C) and D‐serine (D) in 4‐weeks and 8‐weeks of age S286L‐TG and wild‐type littermate. Ordinates indicate mean ± SD ( n = 6) of (A) expression levels of GluN2A relative to GAPDH in the plasma membrane fraction (B) expression levels of GluN2B relative to GAPDH in the plasma membrane fraction, (C) basal extracellular L‐glutamate level (μM) and (D) basal extracellular D‐serine level (μM) in the frontal cortex of wild‐type (gray column) and S286L‐TG (blue column). The lower‐side panels in A and B indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to 4‐weeks of age (4 W) and # p < 0.05 relative to wild‐type using two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (A) expression of GluN2A ( F age [1, 20] = 46.7 [ p < 0.05], F genotype [1, 20] = 5.34 [ p < 0.05], F age*genotype [1, 20] = 1.1 [ p > 0.05]), (B) expression of GluN2B ( F age [1, 20] = 22.4 [ p < 0.05], F genotype [1, 20] = 8.3 [ p < 0.05], F age*genotype [1, 20] = 2.0 [ p > 0.05]), (C) L‐glutamate level ( F age [1, 20] = 3.2 [ p > 0.05], F genotype [1, 20] = 21.2 [ p < 0.05], F age*genotype [1, 20] = 1.9 [ p > 0.05]) and (D) D‐serine level ( F age [1, 20] = 8.4 [ p < 0.05], F genotype [1, 20] = 21.6 [ p < 0.05], F age*genotype [1, 20] = 2.8 [ p > 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Clinical Proteomics, Membrane, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic administration of probenecid, MK‐801, memantine, and FP802 on expression of GluN2A and GluN2B in S286L‐TG and wild‐type littermates. All rats were chronically administered by vehicle (control), probenecid (PBN: 100 mg/kg/day), MK‐801 (0.1 mg/kg/day), memantine (MEM: 10 mg/kg/day) and FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of expression levels of GluN2A (A1‐A4) and GluN2B (B1‐B4) relative to GAPDH in wild‐type (A1‐A2, B1‐B2) and S286L‐TG (A3‐A4, B3‐B4). The right‐side panels indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control using one‐way ANOVA with Scheffe's post hoc test. F ‐values regarding effects of probenecid and MK‐801 on GluN2A expression in wild‐type (A1) ( F [2, 15] = 7.8 [ p < 0.05]), GluN2A in S286L (A3) ( F [2, 15] = 19.4 [ p < 0.05]), GluN2B in wild‐type (B1) ( F [2, 15] = 12.1 [ p < 0.05]) and GluN2B in S286L‐TG (B3) ( F [2, 15] = 18.2 [ p < 0.05]). F ‐values regarding effects of memantine and FP802 on GluN2A in wild‐type (A2) ( F [2, 15] = 0.4 [ p > 0.05]), GluN2A in S286L‐TG (A4) ( F [2, 15] = 7.1 [ p < 0.05]), GluN2B in wild‐type (B2) ( F [2, 15] = 0.2 [ p > 0.05]) and GluN2B in S286L‐TG (B4) ( F [2, 15] = 4.8 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot

Journal: Pharmacology Research & Perspectives

Article Title: Combined Inhibition of TRPM 4/ NMDA Receptor Complex and Extrasynaptic NMDA Receptors Is Candidate Therapeutic Target for Suppression of Epileptic Seizures and Improvement of Cognitive Impairments

doi: 10.1002/prp2.70256

Figure Lengend Snippet: Effects of chronic combined administration of memantine with FP802 on ADSHE seizure frequency (A), sucrose preference (B), expression of GluN2A (C1) and GluN2B (C2), and basal extracellular levels of L‐glutamate (D) and D‐serine (E) in S286L‐TG and wild‐type littermate. All rats were chronically administered by vehicle (control) and combined of memantine (MEM: 10 mg/kg/day) with FP802 (40 mg/kg/day) for 2‐weeks (from 6‐weeks to 8‐weeks of age). Ordinates indicate mean ± SD ( n = 6) of (A) ADSHE seizure frequency (count h −1 ), (B) consumption of sucrose preference (%), (C1) expression levels of GluN2A relative to GAPDH, (C2) expression levels of GluN2B relative to GAPDH, (D) basal extracellular L‐glutamate level (μM) and (E) basal extracellular D‐serine level (μM). The right‐side panels in C1‐C2 indicate pseudo‐gel images of capillary immunoblotting. Circles indicate the values of each individual rat. * p < 0.05, relative to control and # p < 0.05 relative to wild‐type using student T ‐test or one‐way or two‐way ANOVA with Scheffe's post hoc test. F ‐values were in (B) sucrose preference: MEM ( F memantine+FP802 [1, 20] = 21.3 [ p < 0.05], F genotype [1, 20] = 5.1 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 5.3 [ p < 0.05]), (D) L‐glutamate level: ( F memantine+FP802 [1, 20] = 5.3 [ p < 0.05], F genotype [1, 20] = 15.9 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 4.8 [ p < 0.05]), (E) D‐serine level: ( F memantine+FP802 [1, 20] = 7.6 [ p < 0.05], F genotype [1, 20] = 22.4 [ p < 0.05], F rmemantine+FP802*genotype [1, 20] = 10.4 [ p < 0.05]).

Article Snippet: Primary antibodies against GAPDH (NB300‐327, RRID:AB_10001915, 1:300; Novus Biologicals, Littleton, CO, USA), GluN2A (PPS012, RRID:AB_2112297, 1:100, R&D Systems, Minneapolis, MN, USA), GluN2B (PPS013, RRID:AB_562667, 1:100, R&D Systems), cAMP response element binding protein (CREB) (#4820, 1:50, Cell Signaling Technology, Danvers, MA, USA), and pCREB (#9198, 1:50, Cell Signaling) were used.

Techniques: Expressing, Control, Western Blot