Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Representative images of clustering of NR2B in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. Higher magnification views are shown of the dendritic branches studded with numerous clusters enclosed in white boxes. Scale bar, 50 µm. (B) and (C) show the quantification of (A). The numbers of NR2B clusters were 25.1±1.0 (control, n = 37), 25.5±1.0 (NBQX+nimodipine, n = 36), and 25.7±1.0 (Zn+NBQX+nimodipine, n = 37) per 100 µm dendrite length. The mean intensity of NR2B clusters was 122.4±3.3 (control, n = 25), 133.0±4.5 (NBQX+nimodipine, n = 25) and 131.3±3.6 (Zn+NBQX+nimodipine, n = 23). (D) Representative NR2B currents in response to co-application with the agonist plus NVP-AAM077. (E) Statistics of NR2B currents. Averaged peak currents were 342.7±71.7 pA (control, n = 38), 327.0±67.3 pA (NBQX+nimodipine, n = 20) and 320.0±48.5 pA (Zn+NBQX+nimodipine, n = 20).

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques:

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Hippocampal neurons derived from control, NBQX+nimodipine and Zn+NBQX+nimodipine groups were incubated in 35 µg/ml CHX. Total expressions of NR1, NR2A and NR2B were measured at 0 hr, 12 hr or 18 hr in the CHX treatment. (B) shows the quantification of (A). (C) Surface NR1, NR2A and NR2B derived from cultured hippocampal neurons were isolated by biotinylation assay and detected by anti-NR1, anti-NR2A and anti-NR2B antibodies. Surface NR1 and NR2A, but not NR2B, were notably lower in the Zn+NBQX+nimodipine group, while total NR1, NR2A and NR2B did not change. (D) Quantifications of (C). Percentage changes of surface signal intensities versus control were: NR1, 97.6±13.8 (NBQX+nimodipine) and 69.4±8.0 (Zn+NBQX+nimodipine); NR2A, 87.7±4.9 (NBQX+nimodipine) and 57.0±14.3 (Zn+NBQX+nimodipine); and NR2B, 99.0±19.0 (NBQX+nimodipine) and 101.0±21.8 (Zn+NBQX+nimodipine). All experiments were performed at least 3 times (N > = 3). *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Derivative Assay, Incubation, Cell Culture, Isolation, Cell Surface Biotinylation Assay

Journal: PLoS ONE

Article Title: Chronic Zinc Exposure Decreases the Surface Expression of NR2A-Containing NMDA Receptors in Cultured Hippocampal Neurons

doi: 10.1371/journal.pone.0046012

Figure Lengend Snippet: (A) Lysates were immunoprecipitated with anti-PSD-95 antibody in control, NBQX+nimodipine and Zn+NBQX+nimodipine groups. The immunoprecipitates were probed with the anti-NR2A (NR2A), anti-NR1 (NR1), anti-NR2B (NR2B), anti-Src (Src) and anti-Fyn (Fyn) antibodies. The Input lane was loaded with total protein (20 µg) derived from cultured hippocampal neurons. (B) Supernatant samples were immunoprecipitated with anti-PSD-95 antibody in different groups, which showed the clearance of PSD95 after Co-IP experiment. All experiments were performed at least 3 times (N > = 3) and the summarized data are shown in (C) and (D). Y axes in (C) and (D) represent the percentage change of each co-precipitated protein (labeled on X axes) relative to the corresponding control. *, P<0.05.

Article Snippet: Rabbit polyclonal NR2B antibody was from Alomone Labs (Jerusalem, Israel).

Techniques: Immunoprecipitation, Derivative Assay, Cell Culture, Co-Immunoprecipitation Assay, Labeling

Journal: PLoS ONE

Article Title: Inhibition of Glycogen Synthase Kinase-3β Prevents Remifentanil-Induced Hyperalgesia via Regulating the Expression and Function of Spinal N-Methyl-D-Aspartate Receptors In Vivo and Vitro

doi: 10.1371/journal.pone.0077790

Figure Lengend Snippet: Western blot for membrane NR1, NR2A and NR2B subunit was performed on rat spinal cord dorsal horn neuron (A). Epidermal growth factor receptor (EGFR) was used as the loading control. Pooled densitometric results for NR1, NR2A and NR2B, with the band intensity of C group assigned the value of 1. Remifentanil induced significant increases of both membrane NR1 and NR2B, but had no effect on membrane protein level of NR2A. GSK-3β inhibitor TDZD-8 prevented the changes of membrane NR1 and NR2B. n = 5 for each group, compared with C group, * P < 0.05, ANOVA (B). The expression of total NR1, NR2A and NR2B protein in spinal dorsal horn was tested by Western blot (C). β-actin was used as the loading control. Densitometry measurements from 5 groups were pooled and the band intensity of C group was assigned a value of 1. Remifentanil increased the total protein level of NR1 and NR2B, but had no effect on total protein level of NR2A. TDZD-8 prevented the changes of total protein expression of NR1 and NR2B (D). n = 5 for each group, compared with C group, * P < 0.05, ANOVA.

Article Snippet: The membranes were blocked with 5% nonfat milk in Tris-Tween buffer saline for 1 h (TBST; 50 mM Tris-HCl, 154 mM NaCl, and 0.05% Tween 20, pH 7.4), incubated overnight at 4 °C with polyclonal rabbit antibodies against rat NR1, NR2A, NR2B (all 1:300 dilution in 5% nonfat milk in TBST, Chemicon, USA) or rabbit anti-rat GSK-3β and phosphorylated (ser9) GSK-3β antibodies (all 1:1000 dilution in 5% nonfat milk in TBST, Cell Signaling Technology, USA), then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (1:2, 000 in 5% nonfat milk in TBST, Jackson Immuno Research, USA) for 1 h. Membrane bound secondary antibodies were detected using Chemiluminescence plus reagent (Perkin Elmer Life and Analytical Sciences, USA) and visualized using a chemiluminescence imaging system (Syngene, Cambridge, UK).

Techniques: Western Blot, Expressing

Journal: Drug Design, Development and Therapy

Article Title: Atomoxetine affects transcription/translation of the NMDA receptor and the norepinephrine transporter in the rat brain – an in vivo study

doi: 10.2147/DDDT.S50448

Figure Lengend Snippet: Atomoxetine induced changes on Grin2B messenger (m)RNA and NR2B levels. Notes: Male adolescent rats were treated for 21 days (postnatal days 21–41) with atomoxetine hydrochloride (3 mg/kg, intraperitoneal [ip]) or saline (0.9%, ip). The striatum (STR), mesencephalon (MES), and hippocampus (HC) of the early treatment group were analyzed 24 hours after the last ip application, whereas the brains of rats assigned to the late treatment group were probed after a treatment-free period of 2 months. ( A ) Glutamate receptor ionotropic N-methyl-D-aspartate 2B (Grin2B) expression was probed in the STR, MES, and HC. Bar diagrams depict mean values of quantitative reverse transcription polymerase chain reaction (qRT-PCR) measurement. ( B ) Bar diagrams depict mean values obtained by densitometric quantification of the ( C ) respective Western blots detecting the glutamate receptor ionotropic N -methyl- D -aspartate 2B (NR2B) at a molecular weight of ∼170 kDa. Controls were male age-matched saline-treated rats. All data are presented as ± standard error of the mean; early treatment group n=7; late treatment group n=8; * P <0.05; ** P <0.01.

Article Snippet: The following primary antibodies were used: rabbit polyclonal antibody against the NET (SAB2102224; Sigma-Aldrich) at a 1:1,000 dilution, rabbit polyclonal antibody against the NMDAR subunit NR1 (SAB4300405; Sigma-Aldrich) at a dilution of 1:1,000, rabbit polyclonal antibody against the NMDAR subunit NR2A (M-264; Sigma-Aldrich) at 1:1,000, rabbit polyclonal antibody against the NMDAR subunit NR2B (AP08705PU-N; Acris Antibodies GmbH, Herford, Germany) at 1:1,000, mouse monoclonal antibody against synaptophysin (Syp) (S 5768; Sigma-Aldrich) at 1:1,000, and rabbit polyclonal antibody against the synaptosomal-associated protein 25 kDa (Snap25) (ab41455; Abcam, Cambridge, UK) 1:2,000.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Molecular Weight