Journal: Journal of Biomedical Science

Article Title: Dual regulation by ethanol of the inhibitory effects of ketamine on spinal NMDA-induced pressor responses in rats

doi: 10.1186/1423-0127-19-11

Figure Lengend Snippet: (a) Left panel: western blot analysis of the levels of phosphoserine 896 on GluN1 subunit (pGluN1-serine 896), phosphoserine 897 on GluN1 subunit (pGluN1-serine 897), and GluN1 subunit (GluN1) in lateral horn regions of T7-T9 segments of rat spinal cord without (control) and at 10 min and 30 min after intravenous injection of ethanol (EtOH, 0.025 g) . The percentage changes in the ratio of pGluN1 (serine 896 and serine 897) to GluN1 are shown in the right panel. The ratio of pGluN1 to GluN1 in rats without administration of ethanol is taken as control (100%). Panels (b) and (c) are similar to panel (a) except that 0.16 g and 0.32 g ethanol were injected, respectively. Values denote mean + SEM from 4-5 separate experiments. *Statistically significant difference from control analyzed by one-way ANOVA followed by Newman- Keuls post-test.

Article Snippet: After NC membrane blocking and washing, blots were probed with primary antibody, rabbit anti-GluN1 polyclonal antibody (1:800, Upstate Biotechnology Inc., Lake Placid, NY, USA) and rabbit anti-pGluN1 antisera (serine 896, 1:1500 and serine 897, 1:1500, Upstate Biotechnology Inc.) in TBS-T containing 5% skimmed milk powder overnight at 4°C on a 2D shaker.

Techniques: Western Blot, Injection

Journal: Journal of Biomedical Science

Article Title: Dual regulation by ethanol of the inhibitory effects of ketamine on spinal NMDA-induced pressor responses in rats

doi: 10.1186/1423-0127-19-11

Figure Lengend Snippet: (a) Left panel: Western blot analysis of the levels of phosphoserine 897 on the GluN1 subunit (pGluN1-serine 897, top) and GluN1 subunit (GluN1, bottom) in lateral horn regions of spinal cord at 10 min after intrathecal injection of saline (control) and cAMPS-Sp (0.5 and 5 nmol) . The percentage changes in the ratio of pGluN1-serine 897 to GluN1 are shown in the right panel. Values denote mean + SEM from three separate experiments. *Significant difference from control (100%). (b) Representative recordings of changes in blood pressure induced by an intrathecal injection of NMDA (2 nmol) before (control) and at 10 min and 40 min after intravenous injection of ketamine (2 mg/kg, left panel; 4 mg/kg, right panel) following pretreatment with cAMPS-Sp (5 nmol); cAMPS-Sp was applied intrathecally 10 min before the administration of ketamine. (c) Bar graphs show percentage changes in NMDA-induced increases in MAP 10 min and 40 min after intravenous injection of ketamine (2 mg/kg, left part; 4 mg/kg, right part) following intrathecal pretreatment with various doses of cAMPS-Sp. *Statistically significant difference from ketamine alone group (without pretreatment with cAMPS-Sp) analyzed using two-way ANOVA followed by Bonferroni post-test.

Article Snippet: After NC membrane blocking and washing, blots were probed with primary antibody, rabbit anti-GluN1 polyclonal antibody (1:800, Upstate Biotechnology Inc., Lake Placid, NY, USA) and rabbit anti-pGluN1 antisera (serine 896, 1:1500 and serine 897, 1:1500, Upstate Biotechnology Inc.) in TBS-T containing 5% skimmed milk powder overnight at 4°C on a 2D shaker.

Techniques: Western Blot, Injection

Journal: Journal of Affective Disorders Reports

Article Title: Long-term cyclosporine A treatment promotes anxiety-like behavior: Possible relation with glutamate signaling in rat hippocampus

doi: 10.1016/j.jadr.2022.100394

Figure Lengend Snippet: Fig. 4. Western blot analysis of NMDA receptors in the hippocampus of control and CsA-treated rats. (A) Representative images of the levels of total (pan) GluN2, GluN2A, GluN2B, p-GluN1 (Ser897) and total GluN1. (B-F) Respective quantifications of immunodetections normalized to β-actin and presented as a percentage of the control mean. Data represent the mean ± SEM. Statistics: ** *p ≤0.001 as determined by Student’s t-test; n = 6.

Article Snippet: The utilized primary antibodies and respective dilutions were as follows: anti-AMPAR (1:500, #13185 Cell Signaling Technology), phosphorylated AMPAR (pAMPAR-S831) (1:1000, A4352-Sigma–Aldrich), pAMPAR (S845) (1:1000, #8084 – Cell Signaling Technology), GluN1 (1:750, #G8913 – Sigma–Aldrich), pan-GluN2 (1:750, 244-0P – SYSY Synaptic Systems), GluN2A (1:1000, #4205 - Cell Signaling Technology), GluN2B (1:1000, #4207 - Cell Signaling Technology), pGluN1 (Ser897) (1:500, 3385S – Cell Signaling Technology), nNOS (1:500, SC648 – Santa Cruz Biotechnology), and iNOS (1:750, SC7271 – Santa Cruz Biotechnology). β-actin antibody (1:5000, A5441 – Sigma-Aldrich) was used as a loading control in the experiments.

Techniques: Western Blot, Control

Journal: The European journal of neuroscience

Article Title: Ghrelin receptor activity amplifies hippocampal NMDA receptor-mediated postsynaptic currents and increases phosphorylation of GluN1 subunit at Ser 896 and Ser 897

doi: 10.1111/ejn.13107

Figure Lengend Snippet: pGluN1 immunoreactivity. A. Representative pGluN1 signals (A1) were manually selected (red circles in A2) and processed for quantitative analysis as described in Methods. A2 shares the same calibration with A1. B-C. pGluN1 immunoreactivity in response to ascending doses of ghrelin in acutely-prepared rat hippocampal slices (B) and in response to GHSR1a antagonists, D-Lys3-GHSR6 (1 μM) and Substance P analogue (10 μM) with or without ghrelin (100 nM) (C). D-E. pGluN1 immunoreactivity in response to ascending doses of ghrelin in cultured rat hippocampal slices (D) and in response to GHSR1a antagonists, D-Lys3-GHSR6 (1 μM) and Substance P analogue (10 μM) with or without ghrelin (100 nM) (E). F-G. pGluN1 immunoreactivity in response to ascending doses of non-peptide agonist for GHSR1a, L-692,585 in cultured rat hippocampal slices (F) and in response to GHSR1a antagonists with or without L-692,585 (100 nM) (G). Photomicrographs in C1 (control), C2 (ghrelin), C3 (ghrelin and D-Lys3-GHRP6), and C4 (ghrelin and Substance P analogue) were taken from Experiment C. Photomicrographs in E1 (control), E2 (ghrelin), E3 (ghrelin and D-Lys3-GHRP6), and E4 (ghrelin and Substance P analogue) were taken from Experiment E. Photomicrographs in G1 (control), G2 (L-629,585), G3 (L-629,585 and D-Lys3-GHRP6) and G4 (L-629,585 and Substance P analogue) were taken from Experiment G. H. Representative pGluN1 immunoreactivity in control and ghrelin in wild-type (GHSR1a +/+), heterozygous (GHSR1a +/-), and homozygous (GHSR1a -/-) knock-out mice (H1). The result was quantified and summarized in H2. Calibration in C1 (5 μm) is shared by all photomicrographs except for A1 and A2. Data are mean ± SEM.

Article Snippet: Immunohistochemistry Slices were immersion-fixed with 4% paraformaldehyde in 1 M phosphate buffered saline (PBS), rinsed, treated with 0.2% Triton-X100 and 10% BSA for 1 hour, and incubated in a rabbit polyclonal anti-GHSR1a (1:200, Phoenix Pharmaceutical, Burlingame, CA; or 1:50, Santa Cruz Biotechnology, Santa Cruz, CA), or a goat polyclonal anti-pGluN1 (phosphorylated GluN1 subunit at Ser 896/897, 1:100, Santa Cruz Biotechnology, Santa Cruz, CA) for 48 hours at 4°C with gentle agitation, which was followed by Alexa 488-conjugated secondary antibody (1:200, Life Technologies, Grand Island, NY) for 1 hour at room temperature.

Techniques: Cell Culture, Knock-Out

Journal: Scientific Reports

Article Title: CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice

doi: 10.1038/srep24250

Figure Lengend Snippet: ( A ) WB analysis of the levels of the indicated proteins in whole hippocampal homogenates (Total) and PSD fractions obtained from Calhm1 +/+ and Calhm1 −/− mice. ( B ) Primary neurons isolated from Calhm1 +/+ and Calhm1 −/− mice were challenged with the calcium add-back condition (CaAB) or not (Basal). Cell extracts were analyzed by WB for the indicated proteins. Representative results from 4 independent experiments are depicted. ( C,D ) Densitometric analysis and quantification of the ratio for phospho-Ser-897 GluN1 over total GluN1 (pGluN1/GluN1), ( C ) and for phospho-Ser-845 GluA1 over actin (pGluA1/actin), ( D ) from Calhm1 +/+ and Calhm1 −/− primary neurons treated as in ( B ). au, arbitrary units ( n = 6; * P < 0.05; ** P < 0.01; t test). ( E ) WB analysis of the levels of the indicated proteins in Calhm1 +/+ primary neurons pretreated for 30 min with H89 (10 μM) and then challenged with CaAB, as in ( B ). ( F,G ) Ratio for phospho-Ser-897 GluN1 over total GluN1 (pGluN1/GluN1), ( F ) and for phospho-Ser-845 GluA1 over actin (pGluA1/actin), ( G ) from LTP-stimulated hippocampal slices ( n = 10–12; * P < 0.05; t test). ( H ) WB analysis of the levels of the indicated proteins in Calhm1 +/+ and Calhm1 −/− primary neurons challenged with CaAB, as in ( B ), in the absence (Ctrl) or presence of glutamate stimulation (Glu, 20 μM, 1 h incubation for c-Fos, 4 h for c/EBPβ).

Article Snippet: Antibodies directed against ERK1/2, phospho-ERK1/2 (pERK1/2, Thr-202/Tyr-204), pGluN1 (Ser-897), GluN2A, GluN2B, pGluA1 (Ser-845), and c-Fos were from Cell Signaling Technology.

Techniques: Isolation, Incubation