Journal: eLife

Article Title: Molecular basis of interactions between CaMKII and α-actinin-2 that underlie dendritic spine enlargement

doi: 10.7554/eLife.85008

Figure Lengend Snippet:

Article Snippet: Recombinant DNA reagent , pEGFP-NR2B vector , Stefano Vicini/Addgene , RRID: Addgene_17925 , .

Techniques: In Situ, Concentration Assay, Recombinant, Construct, Plasmid Preparation, Protease Inhibitor, Software

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: Antioxidants (Basel, Switzerland)

Article Title: The Application of the Neuroprotective and Potential Antioxidant Effect of Ergotamine Mediated by Targeting N-Methyl-D-Aspartate Receptors.

doi: 10.3390/antiox11081471

Figure Lengend Snippet: Figure 2. (A) The chemical structure of ergotamine. (B) The H2O-injected oocytes did not cause any change with the treatment of 100 µM glutamate (n = 6–8 oocytes from four different frogs). (C–F) Glutamate induced inward currents with or without ergotamine (30 µM and 10 µM). For each subunit of NMDARs, the responses after treating with either glutamate (100 µM) alone or together with ergotamine (30 and 10 µM). Voltage clamp recording was conducted at a holding potential of −80 mV. The coapplication of ergotamine with glutamate resulted in modulation of the recombinant receptors (C) NR1a/NR2A, (D) NR1a/NR2B, (E) NR1a/NR2C, and (F) NR1a/NR2D, which in turn reduced glutamate-evoked inward current in a reversible manner (n = 6–8 oocytes from four different frogs).

Article Snippet: The mouse NMDAR subunit cDNAs included the NR1 (GenBank accession number: MR225704), NR2A (MR227135), NR2B (MR227077), NR2C (MR222676), and NR2D (MR220972) subunits, which were purchased from OriGene (Rockville, MD, USA).

Techniques: Injection, Recombinant

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Regional expression changes in GluN2A, GluN2B, and GABA A R in WT and AD mice with aging. (A) Representative immunoblots showing GluN2A, GluN2B, and GABA A R expression across the hippocampus, prefrontal cortex, cortex, midbrain, and cerebellum in young and old WT mice. GluN2A expression remained largely stable with aging, except for an increase in the prefrontal cortex. GluN2B expression decreased with age across most regions, while GABA A R expression was reduced in the hippocampus and cortex in old WT mice. (B) Representative immunoblots from young and old AD mice showing GluN2A, GluN2B, and GABA A R levels across the same regions. GluN2A expression increased with aging in nearly all regions, while GluN2B and GABA A R levels decreased broadly.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Comparative Analysis of GluN2A, GluN2B, and GABA A R Receptor Expression in Young and Old WT vs AD Mice. (A,B) Western blot analysis comparing GluN2A levels between WT and AD mice showed increased GluN2A expression in hippocampal and cortical regions of old WT mice compared to AD and a slight decrease in the prefrontal cortex of young AD mice. (C,D) No significant change region-wide between ages, except for an observed decrease in the prefrontal cortex of young AD mice. (E,F) Downregulated GABA A R expression is seen in old AD mice across all regions compared to WT. For each brain region, n = 4–5 mice per group. Bar graphs depict the mean ± SD normalized to β-actin. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Increased extrasynaptic localization of GluN2B subunits with disease progression in AD mice and comparative analysis of ES-GluN2B and synaptic GluN2B expression in midbrain and cortex between WT and AD mice. (A,B) Immunoblot analysis showing significant increases in ES-GluN2B levels across brain regions of AD mice. Quantification reveals a marked increase in ES-GluN2B expression in these regions, indicating a shift from synaptic to extrasynaptic localization with disease. No significant changes in the cerebellum of AD mice. (C,D) Results demonstrate significant increases in ES-GluN2B levels in old AD mice compared to old WT mice, emphasizing the disease-dependent shift toward extrasynaptic localization, which becomes more pronounced with aging in these brain regions. Bar graphs represent the mean ± SD of ES-GluN2B levels normalized to Synaptic GluN2B. For each brain region, n = 4–5 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Biomarker Discovery, Expressing, Western Blot

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Region-specific increases in GluN2B phosphorylation and casein kinase IIα (CK2α) expression in AD brain regions. (A,B) Immunoblot and quantification of CK2α protein levels across brain regions (hippocampus, prefrontal cortex, cortex, and midbrain) of aged AD mice compared to aged WT mice. CK2α expression significantly increases with age in AD mice, whereas WT mice exhibit stable or reduced levels. n = 4–5 per group. (C,D) Western blot and quantification showing significant increases in phosphorylation of GluN2B at Ser1480 (pGluN2B) across brain regions (hippocampus, prefrontal cortex, cortex, and midbrain) of aged AD mice compared to aged WT mice. Old AD mice also showed higher pGluN2B levels than their WT counterparts in all regions. The ratio of pGluN2B to total GluN2B is markedly elevated in old AD mice, supporting increased extrasynaptic signaling activity with disease progression. n = 4–5 per group. All bar graphs depict mean ± SD of protein levels normalized to loading controls (GluN2B or β-actin). * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Phospho-proteomics, Expressing, Western Blot, Activity Assay, Biomarker Discovery

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Region-specific expression of polysialylation enzymes ST8Sia2 and ST8Sia4 in AD and normal aging. (A,B) Western blot images and quantitative analysis of ST8Sia4 protein expression in the hippocampus, prefrontal cortex, cortex, and midbrain of young and aged WT and AD mice. ST8Sia4 expression significantly increased with age in WT mice but declined in aged AD mice across all regions, notably in the prefrontal cortex and cortex. Similarly, ST8Sia4 showed a significant decrease in aged AD mice compared to aged WT mice. (C,D) ST8Sia2 protein levels showed no significant regional differences. No significant changes were observed across age groups region-wide. (E) Parental IMR32 cells are unmodified. In gRNA-ST conditions, IMR32 cells are transfected with plasmid DNA that expresses dCas9-VP64 and the guide RNA targeting the ST8sia4 promoter, enabling CRISPR-mediated transcriptional activation of ST8sia4. (F) Western blot analysis revealed significantly higher GluN2B phosphorylation at Ser1480 in parental cells compared to PSA–NCAM–overexpressing cells (gRNA-ST), (G) increased PSA-NCAM expression in gRNA-ST cells, (H) with no significant difference in CKIIα levels. Bar graphs depict mean ± SD of protein levels normalized to loading controls (GluN2B or β-actin), n = 3 per group. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Expressing, Western Blot, Transfection, Plasmid Preparation, CRISPR, Activation Assay, Phospho-proteomics

Journal: Frontiers in Neuroscience

Article Title: Spatiotemporal differential regulation of extrasynaptic GluN2B receptor subunits and PSA-NCAM in brain aging and Alzheimer’s disease

doi: 10.3389/fnins.2025.1649625

Figure Lengend Snippet: Summary of NMDA-GABA receptor-mediated E/I Balance in Normal Aging and AD. The figure illustrates the shift in E/I balance during normal aging (top) and Alzheimer’s disease (bottom). In normal aging, the balance is maintained by decreased total GluN2B level and increasing GluN2A subunit expression. In contrast, AD shows a significant decrease in GluN2B expression and an upregulation of GluN2A, alongside a marked reduction in GABA A R expression, reflecting diminished inhibitory signaling and overall E/I imbalance. Additionally, increased ES-GluN2B localization further contributes to synaptic dysfunction and excitotoxicity in AD.

Article Snippet: To assess the relationship between PSA-NCAM expression and GluN2B phosphorylation, both the engineered ST8Sia4-overexpressing IMR-32 cells and parental IMR-32 cells were co-transfected with GluN1 (SC308819, Origene Technologies Inc., Rockville, MD), GluN2B (SC119642, Origene Technologies Inc., Rockville, MD), and pMaxGFP plasmids at a 2:2:1 ratio.

Techniques: Expressing