Journal: Neural Plasticity

Article Title: Seizure-Induced Regulations of Amyloid- β , STEP 61 , and STEP 61 Substrates Involved in Hippocampal Synaptic Plasticity

doi: 10.1155/2016/2123748

Figure Lengend Snippet: A single ECS and chronic ECS differently alter Tyr 204/187 -phosphorylation of ERK1/2 in the hippocampus. Immunoblot analysis for the phosphorylation of ERK1/2 at Tyr 204/187 (Y 204/187 ) and total ERK1/2 expression in the hippocampal crude membrane (P2) fraction following a single ECS ( n = 5 rats per time point) (a) and chronic ECS ( n = 6 rats per time point) (b). The ratio of the Tyr 204/187 -phosphorylated ERK1/2 band intensity over the β -actin band intensity (top graphs) and the ratio of total ERK1/2 band intensity over the β -actin band intensity (bottom graphs) were calculated per each time point and normalized to that of “no seizure” (NS) sham group. Data shown represent the mean band intensity ± SEM. (a) A single ECS transiently decreases the level of Tyr 204/187 -phosphorylated ERK1/2 in the hippocampus at 48 h ( ### p < 0.005, t -test) and total ERK1/2 expression at 72–96 h ( ∗ p < 0.05) following a single ECS. (b) Chronic ECS significantly increases the level of Tyr 204/187 -phosphorylated ERK1/2 at 0 h following chronic ECS ( ∗ p < 0.05) but has no effect on total GluN2B expression in the hippocampus.

Article Snippet: Phosphorylation site specific antibodies used include anti-GluN2B-pTyr 1472 which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr 202 /Tyr 204 which recognizes phosphorylated Thr 202 /Tyr 204 of ERK1 and Thr 185 /Tyr 187 of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr 869 , Tyr 873 , and Tyr 876 (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY 876 which recognizes phosphorylated Tyr 876 of GluA2 (#4027S, Cell Signaling).

Techniques: Phospho-proteomics, Western Blot, Expressing, Membrane

Journal: Neural Plasticity

Article Title: Seizure-Induced Regulations of Amyloid- β , STEP 61 , and STEP 61 Substrates Involved in Hippocampal Synaptic Plasticity

doi: 10.1155/2016/2123748

Figure Lengend Snippet: A single ECS but not chronic ECS transiently decreases the level of Tyr 1472 -phosphorylated GluN2B in the hippocampus. Immunoblot analysis for the phosphorylation of GluN2B at Tyr 1472 (Y 1472 ) and total GluN2B expression in the hippocampal crude membrane (P2) fraction following a single ECS ( n = 5 rats per time point) (a) and chronic ECS ( n = 6 rats per time point) (b). The ratio of the Tyr 1472 -phosphorylated GluN2B band intensity over the β -actin band intensity (top graphs) and the ratio of total GluN2B band intensity over the β -actin band intensity (bottom graphs) were calculated per each time point and normalized to that of “no seizure” (NS) sham group. Data shown represent the mean band intensity ± SEM. (a) A single ECS transiently decreases the level of Tyr 1472 -phosphorylated GluN2B in the hippocampus at 48 h ( ∗∗∗ p < 0.005) and 72 h ( ∗ p < 0.05) following a single ECS. (b) Chronic ECS significantly decreases total GluN2B expression over the time course of 96 h in the hippocampus ( ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.005).

Article Snippet: Phosphorylation site specific antibodies used include anti-GluN2B-pTyr 1472 which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr 202 /Tyr 204 which recognizes phosphorylated Thr 202 /Tyr 204 of ERK1 and Thr 185 /Tyr 187 of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr 869 , Tyr 873 , and Tyr 876 (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY 876 which recognizes phosphorylated Tyr 876 of GluA2 (#4027S, Cell Signaling).

Techniques: Western Blot, Phospho-proteomics, Expressing, Membrane

Journal: Neural Plasticity

Article Title: Seizure-Induced Regulations of Amyloid- β , STEP 61 , and STEP 61 Substrates Involved in Hippocampal Synaptic Plasticity

doi: 10.1155/2016/2123748

Figure Lengend Snippet: Model by which seizure-induced changes in A β , STEP 61 , and Tyr-phosphorylation of STEP 61 lead to synaptic weakening in the hippocampus. A single ECS increases STEP 61 expression and decreases Tyr-phosphorylation of NMDAR subunit GluN2B and ERK1/2 in the hippocampus at 48 h time point, leading to synaptic weakening via NMDAR internalization and ERK1/2 inactivation. A delayed decrease in ERK1/2 expression as well as a delayed enhancement of APP and A β expression at 72–96 h following a single ECS maintains this synaptic weakening. Chronic ECS-induced increase in APP expression and A β production as well as persistent decrease in total GluN2B level leads to synaptic weakening.

Article Snippet: Phosphorylation site specific antibodies used include anti-GluN2B-pTyr 1472 which recognizes phosphorylated Tyr-1472 of GluN2B (P1516-1472, PhosphoSolutions), anti-ERK1/2-pThr 202 /Tyr 204 which recognizes phosphorylated Thr 202 /Tyr 204 of ERK1 and Thr 185 /Tyr 187 of ERK2 (#9106, Cell Signaling), anti-GluA2-p3Y which recognizes phosphorylated Tyr 869 , Tyr 873 , and Tyr 876 (3Y) of GluA2 (#3921S, Cell Signaling), and anti-GluA2-pY 876 which recognizes phosphorylated Tyr 876 of GluA2 (#4027S, Cell Signaling).

Techniques: Phospho-proteomics, Expressing

Journal: Theranostics

Article Title: Therapeutic effects of a novel electrode for transcranial direct current stimulation in ischemic stroke mice

doi: 10.7150/thno.90779

Figure Lengend Snippet: Verification of potential signaling pathways of the therapeutic effects of the new HD-tDCS application based on transcriptome analysis. (A) Bar charts of gene expressions for Insig1 , Bag5 , Hdac2 , and U2af2 in the peri-infarct region using qPCR. The expression of these genes was significantly altered by HD-tDCS-FN application compared to that with MCAO. (B) Bar charts of the hs-CRP and TRAIL levels in the peri-infarct region using enzyme-linked immunosorbent assays. The level of TRAIL was significantly reduced in both HD-tDCS-FN groups compared with that in the MCAO group. (C) Representative western blots for NMDA-dependent SREBP signaling and related proteins. The pNR2B(ser1303)/NR2B ratio, preSREBP1, and mSREBP1 levels were significantly decreased in the MCAO+tDCS-FN(cA) group compared with those in the MCAO group. (D) Representative western blots for inflammation-related proteins. COX2 expression was markedly decreased in both MCAO+tDCS-FN groups compared with that in the MCAO group, while only the pAKT(Ser473)/AKT ratio decreased in the MCAO+tDCS-FN(cA) group. β-actin was used as the loading control. All samples were derived from the peri-infarct region (n=5/group). Data are presented as means±SEMs. * P <0.05, ** P <0.01, and *** P <0.001 vs. each group using one-way analysis of variance with Tukey's test. MCAO, middle cerebral artery occlusion; tDCS, transcranial direct current stimulation; FN, new needle-conjugated electrode.

Article Snippet: After blocking in a 5% solution of non-fat milk for 1 h at 20 °C, the membrane was incubated overnight at 4°C with the following primary antibodies: INSIG1 (1:100, ab70784, Abcam), BAG5 (1:500, sc-390832; Santa Cruz Biotechnology, Dallas, TX, USA), NFkB1 (1:500, 51-3500, Thermo Fisher Scientific), HDAC2 (1:500, sc-81599, Santa Cruz Biotechnology), U2AF2 (1:1000, MA5-35532, Thermo Fisher Scientific), SREBP1 (1:500, MA5-16124, Thermo Fisher Scientific), COX2 (1:500, 4842; Cell Signaling Technology, Danvers, MA, USA), NR2A (1:500, NBP2-22404; Novus Biologicals, Centennial, CO, USA), NR2B (1:500, NB100-74475, Novus Biologicals), pNR2B (1:200, 38-7000, Invitrogen), AKT (1:500, 38-7000, Invitrogen), pAKT (1:500, 4058, Cell Signaling Technology), phosphoinositide-3-kinase (PI3K; 1:500, 4257, Cell Signaling Technology), phosphorylated PI3K (pPI3K, 1:500, 4228, Cell Signaling Technology), and ß-actin (1:1000, A2066, Sigma-Aldrich).

Techniques: Protein-Protein interactions, Expressing, Western Blot, Control, Derivative Assay

Journal: Scientific Reports

Article Title: Role of folate receptor α in the partial rejuvenation of dentate gyrus cells: Improvement of cognitive function in 21-month-old aged mice

doi: 10.1038/s41598-024-57095-x

Figure Lengend Snippet: Effect of folate on the expression of NMDA receptor subunit GluN2B and on extracellular matrix organization. Effects of folate on the presence of the NMDA receptor subunit GluN2B in mature hippocampal neurons. ( a,b ) Representative stitched confocal images of GluN2B distribution in the dentate gyrus (DG) of aged wild-type mice treated with vehicle ( a ) or folate ( b ). Higher magnifications (×40 oil immersion objective was used with additional zoom applied) of the molecular cell layer of ( a , b ) (yellow squares) are shown. ( c ) Histogram showing the percentage of area occupied by the GluN2B signal in the molecular layer of the DG. The effect of the absence (dark grey) or presence (light gray) of folate is shown. Treatment with folate induced a significant increase in GluN2B expression in the DG (t = 2.269, df = 8). (d,e) Representative stitched confocal images of Wisteria floribunda agglutinin (WFA, in green), as a marker of perineuronal nets (PNN) in the DG of vehicle-treated and folate-treated mice. The nuclear marker DAPI is shown in blue. Higher magnifications of the molecular cell layer of ( d , e ) (yellow squares) are shown. ( f ) Single-dose treatment with folate resulted in statistically significant changes in PNN density in the DG (t = 2.166, df = 8) (mean ± SEM; *p < 0.05, Student’s t-test). Scale bars, 100 µm ( a,b,d,e ) and 10 µm (higher magnifications).

Article Snippet: Subsequently, brain sections were incubated for 24 h at 4 °C in the same pre-incubation stock solution (PB + Triton + Serum) containing different combinations of the following primary antibodies: ab6326 (Abcam) for CldU; AB2253 (Sigma Aldrich) for Doublecortin; ab8898 (Abcam) for Histone H3 (tri methyl K9); ab78517 (Abcam) for Histone H4 (dimethyl K20, tri methyl K20); TYR1336 Antibody (PhosphoSolution) for NMDA GluN2B Subunit (p1516-1336); ab32423 (Abcam) for brain lipid-binding protein (Blbp); 33D3 (Epigentek) for 5-methylcytosine (5mC); 39791 (Active-Motif) for 5-Hydromethylcytosine (5hmC) antibody; L1516 (Merck) for Lectin from Wisteria floribunda (WFA); ABN78 (Millipore) for NeuN and IF03L (Calbiochem) for GFAP.

Techniques: Expressing, Marker

Journal: Scientific Reports

Article Title: Role of folate receptor α in the partial rejuvenation of dentate gyrus cells: Improvement of cognitive function in 21-month-old aged mice

doi: 10.1038/s41598-024-57095-x

Figure Lengend Snippet: Effect of FRα-binding peptide on the expression of GluN2B expression, and extracellular matrix organization ( a,b ). Representative stitched confocal images of GluN2B distribution in the DG of aged wild-type mice treated with vehicle ( a ) or FRα-binding peptide ( b ). Higher magnifications (×40 oil immersion objective was used with additional zoom applied) of the molecular cell layer of ( a,b ) (yellow squares) are shown. The nuclear marker DAPI is shown in blue. ( c ) Histogram showing the percentage of area occupied by the GluN2B signal in the molecular layer of the DG. The effect of the absence (dark grey) or presence (light gray) of FRα is shown. Treatment with FRα-binding peptide induced a significant increase in GluN2B expression in the DG (t = 2.069, df = 10). (d,e) Representative stitched confocal images of Wisteria floribunda agglutinin (WFA, in green), as a marker of perineuronal nets (PNN) in the DG of mice injected with vehicle or FRα-binding peptide. The nuclear marker DAPI is shown in blue. Higher magnifications of the granular cell layer of ( d,e ) (yellow squares) are shown. ( f ) Single-dose treatment with FRα-binding peptide resulted in statistically significant changes in PNN density in the DG (t = 2.771, df = 10) (mean ± SEM; *p < 0.05, **p < 0.01; Student’s t-test). Scale bar, 100 µm ( a,b,d,e ) and 10 µm (higher magnifications).

Article Snippet: Subsequently, brain sections were incubated for 24 h at 4 °C in the same pre-incubation stock solution (PB + Triton + Serum) containing different combinations of the following primary antibodies: ab6326 (Abcam) for CldU; AB2253 (Sigma Aldrich) for Doublecortin; ab8898 (Abcam) for Histone H3 (tri methyl K9); ab78517 (Abcam) for Histone H4 (dimethyl K20, tri methyl K20); TYR1336 Antibody (PhosphoSolution) for NMDA GluN2B Subunit (p1516-1336); ab32423 (Abcam) for brain lipid-binding protein (Blbp); 33D3 (Epigentek) for 5-methylcytosine (5mC); 39791 (Active-Motif) for 5-Hydromethylcytosine (5hmC) antibody; L1516 (Merck) for Lectin from Wisteria floribunda (WFA); ABN78 (Millipore) for NeuN and IF03L (Calbiochem) for GFAP.

Techniques: Binding Assay, Expressing, Marker, Injection

Journal: Neuropharmacology

Article Title: cAMP signaling through protein kinase A and Epac2 induces substance P release in the rat spinal cord.

doi: 10.1016/j.neuropharm.2021.108533

Figure Lengend Snippet: Fig. 8. Forskolin increases phosphorylation of the NR1 and NR2B subunits of the NMDA receptor. Cultured DRG neurons were incubated for 10 min at 37 ◦C with no addition (control, C), 10 μM forskolin (F), or forskolin +10 μM H89 (F+H). Cells were ho mogenized and extracts electrophoresed on SDS- PAGE gels. A: Membrane blots of the gels were pro bed with antibodies to NR1, p-Ser897-NR1 (pNR1), NR2B, or p-Ser1166- NR2B (pNR2B). B: Band in tensities of pNR1 and pNR2B were divided by the intensities of NR1 and NR2B, respectively, and normalized to the controls. Kurskal-Wallis tests: pNR1 p < 0.0001, pNR2B p < 0.0001. Dunn’s post-hoc tests (uncorrected): *** p < 0.0001 compared to control; † p = 0.028 compared to forskolin.

Article Snippet: Blots were blocked with 3% bovine serum albumin in PierceTM Protein-Free T20 blocking buffer and probed with antibodies (in T20 blocking buffer) to NR1 (1:4000, Abcam, ab109182), p-Ser897-NR1 (1:1000, Abcam, ab52184), NR2B (1:2000, Santa Cruz, sc9057) or pSer1166- NR2B (1:1000, PhosphoSolutions p1516-1166).

Techniques: Phospho-proteomics, Cell Culture, Incubation, Control, SDS Page, Membrane