anti glua1  (Proteintech)

Journal: Frontiers in Cellular Neuroscience

Article Title: Ceftriaxone attenuates Poly I:C–induced neuroinflammation in vitro by modulating glutamate transport, synaptic integrity, and immunometabolic reprogramming

doi: 10.3389/fncel.2025.1684398

Figure Lengend Snippet: Ceftriaxone rescues the reduced colocalization of PSD-95 and CaMKIIα induced by Poly I:C. (A) Representative image used for colocalization analysis. The red box denotes the zoomed-in ROI highlighting a primary dendrite of a single neuron. Scale bars: 20 μm (overview) and 2 μm (zoom). (B,C) Quantification of normalized expression levels at 48 h post-treatment: PSD-95 ( B , red) and CaMKIIα ( C , green). (D) Pearson’s R coefficients for colocalization of CaMKIIα and PSD-95 within primary dendrites. (E) Representative immunocytochemical staining of AMPA-receptor subunits GluA1 and GluA2. Scale bar: 100 μm. (F,G) Normalized expression levels of GluA1 (F) and GluA2 (G) at 48 h post-treatment. Data are presented as mean ± SEM. ** p < 0.01, **** p < 0.0001; ns, not significant.

Article Snippet: The primary antibodies used were CaMKIIα (goat, 1:750, Abcam, ab111890), COX4 (rabbit, 1:500, Synaptic Systems, AB_2620041), Connexin 43 (rabbit, 1:500, Sigma-Aldrich, C6219), EAAT1/GLAST-1/SLC1A3 (rabbit, 1:500, Novus Biologicals, NB100-1869), EAAT2/GLT-1 (rabbit, 1:500, Novus Biologicals, NBP1-20136), GFAP (mouse, 1:1000, Sigma-Aldrich, G3893), GluR1 (GluA1) (guinea pig, 1:400, Alomone Labs, AGP-009), GluR2 (GluA2) (rabbit, 1:400, Alomone Labs, AGC-005), IBA-1 (mouse, 1:1000, Synaptic Systems, 234011), PSD-95 (mouse, 1:750, Novus Biologicals, NB300-556), and MAP2 (chicken, 1:1000, Synaptic Systems, 188006).

Techniques: Expressing, Staining

Journal: Cells

Article Title: Non-Competitive AMPA Receptor Antagonist Perampanel Inhibits Ischemia-Induced Neurodegeneration and Behavioral Deficits in Focal Cortical Pial Vessel Disruption Stroke Model

doi: 10.3390/cells14201628

Figure Lengend Snippet: Effect of perampanel on surface levels of GluA1 and GluA2 in both sides of the hippocampus 72 h post-PVD. Focal ischemia resulted in significant decrease in surface levels of both GluA1 and GluA2 on both sides of the hippocampus, and perampanel did not prevent these effects. Total levels of GluA1 and GluA2 were normalized to the corresponding expression of β-actin, and ratios of surface levels/total protein were calculated and expressed as percentage of sham. ( A ) Representative images of Western blots of surface GluA1 and GluA2 from both ipsilateral and contralateral hippocampal tissue lysate. ( B – E ) Bar charts showing the quantification of surface GluA1, pGluA1-S831, pGluA1-S845, and surface GluA2, respectively, in both ipsilateral and contralateral sides. Sham (black), PVD-vehicle control (red), and PVD-perampanel treated group (blue). Focal cortical ischemia caused a significant decrease in surface expression of GluA1 and GluA2 AMPAR subunits and reduced the ratio of phosphorylated S831 and S845 of GluA1. Perampanel treatment partially restored the levels of GluA1 AMPAR subunits in contralateral hippocampus and increased the pGluA1-S831 and pGluA1-S845 from both the ipsilateral and contralateral hippocampus. N = 4 from different rats for each treatment group. Graphed values show mean ± SEM. Two-way ANOVA was used to analyze group and hemisphere effects. Overall results showed only a significant difference between treatment groups, with no significant effect of hemispheres ipsilateral vs. contralateral within the group or interaction between group and hemispheres. Surface GluA2 expression showed a significant main effect of group ( p = 0.0093), with no significant hemisphere effect ( p = 0.4221) or group × hemisphere interaction ( p = 0.8451). Surface phosphorylated GluA1 at Ser831 ( p -S831) exhibited a significant main effect of group ( p = 0.0001), but no effect of hemisphere ( p = 0.7757) or interaction ( p = 0.3031). Similarly, surface phosphorylated GluA1 at Ser845 ( p -S845) showed a significant group effect ( p = 0.0094), with no significant hemisphere effect ( p = 0.6948) or interaction ( p = 0.6914). Total GluA1 levels also differed significantly between groups ( p = 0.0014), with no significant hemisphere effect ( p = 0.7062) or interaction ( p = 0.2758). Post hoc multiple comparisons significance values: * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

Article Snippet: Membranes were incubated with 5% fat-free milk for 1 h at room temperature to block nonspecific background and then treated with primary antibodies overnight at 4 °C as follows: GluA1 (rabbit mAb, Millipore, Burlington, MA, USA), GluA2 (mouse mAb, Millipore), GFAP (Proteintech, Rosemont, IL, USA, cat # 16825-1-AP; 1:5000), Iba-1 (Invitrogen, Carlsbad, CA, USA, cat # GT10312; 1:500 dilution), inducible nitric oxide synthase (iNOS, Abcam, Cambridge, UK, Cat # ab3523; 1:1000), and neuronal nitric oxide synthase (nNOS, Millipore, Burlington, MA, USA, cat # 07-571-I; 1:1000).

Techniques: Expressing, Western Blot, Control

Journal: Cells

Article Title: Non-Competitive AMPA Receptor Antagonist Perampanel Inhibits Ischemia-Induced Neurodegeneration and Behavioral Deficits in Focal Cortical Pial Vessel Disruption Stroke Model

doi: 10.3390/cells14201628

Figure Lengend Snippet: Rat Gria1 (GluA1) and Gria2 (GluA2) amino acid sequence alignment. Alignment was generated using multiple sequence comparison by log expectation (MUSCLE) using rat Gria1 (GluA1) (Uniprot ID P19490 ) and Gria2 (GluA2) (Uniprot ID P19491 ) sequences. Red stars denote amino acid residues that interact with perampanel as revealed from molecular docking analysis (see ). Amino acid numbers on the right refer to the amino acids of mature protein (excluding the signal sequence in red rectangle). Perampanel binds to a region before transmembrane domain M1 (pre-M1), M1, M3, and M4 regions. Perampanel does not bind to the re-entrant loop M2 domain, the extracellular amino terminal domain (ATD) and ligand binding domain (LBD), or the cytoplasmic domain containing the S831 (PKC site) and S845 (PKA site).

Article Snippet: Membranes were incubated with 5% fat-free milk for 1 h at room temperature to block nonspecific background and then treated with primary antibodies overnight at 4 °C as follows: GluA1 (rabbit mAb, Millipore, Burlington, MA, USA), GluA2 (mouse mAb, Millipore), GFAP (Proteintech, Rosemont, IL, USA, cat # 16825-1-AP; 1:5000), Iba-1 (Invitrogen, Carlsbad, CA, USA, cat # GT10312; 1:500 dilution), inducible nitric oxide synthase (iNOS, Abcam, Cambridge, UK, Cat # ab3523; 1:1000), and neuronal nitric oxide synthase (nNOS, Millipore, Burlington, MA, USA, cat # 07-571-I; 1:1000).

Techniques: Sequencing, Generated, Comparison, Ligand Binding Assay

Journal: Cells

Article Title: Non-Competitive AMPA Receptor Antagonist Perampanel Inhibits Ischemia-Induced Neurodegeneration and Behavioral Deficits in Focal Cortical Pial Vessel Disruption Stroke Model

doi: 10.3390/cells14201628

Figure Lengend Snippet: Structure of Gria1 (GluA1) and Gria2 (GluA2) interacting with perampanel (PER). ( A ) GluA1 structure viewed parallel to the membrane. Each subunit is in different color. The inner and outer sides of the membrane are indicated by parallel bars. ( B , C ) Perampanel is presented in yellow. Close-up views of the binding site in GluA1-PER ( B ) and GluA2-PER ( C ) structures (see text for detail). Hydrogen bonding and hydrophobic interactions with perampanel are described in text. ATD, amino terminal domain; LBD, ligand-binding domain; TMD, transmembrane domain.

Article Snippet: Membranes were incubated with 5% fat-free milk for 1 h at room temperature to block nonspecific background and then treated with primary antibodies overnight at 4 °C as follows: GluA1 (rabbit mAb, Millipore, Burlington, MA, USA), GluA2 (mouse mAb, Millipore), GFAP (Proteintech, Rosemont, IL, USA, cat # 16825-1-AP; 1:5000), Iba-1 (Invitrogen, Carlsbad, CA, USA, cat # GT10312; 1:500 dilution), inducible nitric oxide synthase (iNOS, Abcam, Cambridge, UK, Cat # ab3523; 1:1000), and neuronal nitric oxide synthase (nNOS, Millipore, Burlington, MA, USA, cat # 07-571-I; 1:1000).

Techniques: Membrane, Binding Assay, Ligand Binding Assay

Journal: Cells

Article Title: Non-Competitive AMPA Receptor Antagonist Perampanel Inhibits Ischemia-Induced Neurodegeneration and Behavioral Deficits in Focal Cortical Pial Vessel Disruption Stroke Model

doi: 10.3390/cells14201628

Figure Lengend Snippet: A summary of identified underlying downstream signaling of adenosine A1/A2A receptor crosstalk and regulation of GluA2-lacking AMPA receptors following cerebral ischemia. A1R stimulation leads to clathrin-mediated endocytosis of GluA1 and GluA2 AMPARs via activation of p38-mitogen-activated protein kinase (p38-MAPK), c-Jun N-terminal kinase (JNK), and protein phosphatases PP2A, PP2B, and PP1 [ , , , , ]. Chronic A1R stimulation in ex vivo hypoxia/normoxia ischemia model or a pial vessel disruption (PVD)-induced focal cortical stroke model leads to desensitization of A1R and upregulation of A2AR via casein kinase 2 (CK2) [ , ]. Inhibition of A2AR with istradefylline in PVD-induced stroke model prevents neurodegeneration and neuroinflammation and attenuates behavioral abnormalities . Similarly, inhibition of AMPARs with perampanel in PVD-induced stroke model prevents neurodegeneration and neuroinflammation and significantly reduces LTP and behavioral deficits (this study). Whether combined perampanel and istradefylline treatment produces greater neuroprotection and improved behavioral outcomes in stroke model warrants further investigation. Solid black arrows indicate activation, while purple and black dashed arrows represent endocytosis and desensitization, respectively.

Article Snippet: Membranes were incubated with 5% fat-free milk for 1 h at room temperature to block nonspecific background and then treated with primary antibodies overnight at 4 °C as follows: GluA1 (rabbit mAb, Millipore, Burlington, MA, USA), GluA2 (mouse mAb, Millipore), GFAP (Proteintech, Rosemont, IL, USA, cat # 16825-1-AP; 1:5000), Iba-1 (Invitrogen, Carlsbad, CA, USA, cat # GT10312; 1:500 dilution), inducible nitric oxide synthase (iNOS, Abcam, Cambridge, UK, Cat # ab3523; 1:1000), and neuronal nitric oxide synthase (nNOS, Millipore, Burlington, MA, USA, cat # 07-571-I; 1:1000).

Techniques: Activation Assay, Ex Vivo, Disruption, Inhibition

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Changes in the phosphorylation of mTORC1-mediated proteins and in the expression of synaptic proteins after DEX exposure. Beginning on DIV 10, the neural spheroids were exposed to 100 μM DEX for 5 days. Western blotting and immunofluorescence were performed using each primary antibody (n = 4 biological replicates). (A) Western blotting revealed the levels of phospho-Ser 2448 -mTORC1 (a), phospho- Thr 37/46 -4E-BP1 (b), and phospho-Thr 389 -p70S6K (c). (B) Western blot analysis and representative images of immunoblots of PSD-95 (a) and GluA1 (b) are shown. Synaptic markers: PSD-95 (green) costained with the neuronal marker MAP-2 (red); GluA1 (green) costained with MAP-2 (red). Scale bar: 100 μm. The data are presented as the means ± standard deviations. mTORC1, mechanistic target of rapamycin complex I; DEX, dexamethasone; DIV, days in vitro; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1; PSD-95, postsynaptic density protein-95; GluA1, AMPA receptor subunit glutamate receptor 1; MAP-2, microtubule-associated protein-2; CON, controls. * p < 0.05 or ** p < 0.01 vs. CON.

Article Snippet: GluA1 , Cell Signaling Technology (13185) , AB_2732897 , IF (1:1,000) WB (1:1,000).

Techniques: Phospho-proteomics, Expressing, Western Blot, Immunofluorescence, Marker, In Vitro, Binding Assay

Journal: Clinical Psychopharmacology and Neuroscience

Article Title: Establishment of a Depression Model Using Dexamethasone-treated Three-dimensional Cultured Rat Cortical Cells

doi: 10.9758/cpn.25.1269

Figure Lengend Snippet: Schematic diagram showing the molecular mechanisms underlying DEX-induced impaired neuroplasticity. Exposure to DEX downregulates signaling pathways that affect neuroplasticity, including BDNF, sirtuin 1, and mTORC1 signaling. Activation of the mTORC1 signaling pathway induces the synthesis of synaptic proteins as well as BDNF. Secreted BDNF interacts with its receptor TrkB, further activating mTORC1 signaling via PI3K/Akt and MEK/ERK1/2. Therefore, synaptic plasticity is enhanced. Sirtuin 1 is also involved in the regulation of neuroplasticity. The original illustration was created using BioRender (biorender.com). Akt, protein kinase B; AMPA, α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; BDNF, brain-derived neurotrophic factor; DEX, dexamethasone; ERK1/2, extracellular signal-regulated kinase 1/2; GluA1, AMPA receptor subunit glutamate receptor 1; MEK, mitogen‑activated protein kinase; mTORC1, mechanistic target of rapamycin complex I; p70S6K, p70S6 kinase; PI3K, phosphatidyl inositol-3 kinase; PSD-95, postsynaptic density protein-95; TrKB, tropomyosin receptor kinase B; 4E-BP1, eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1.

Article Snippet: GluA1 , Cell Signaling Technology (13185) , AB_2732897 , IF (1:1,000) WB (1:1,000).

Techniques: Protein-Protein interactions, Activation Assay, Derivative Assay, Binding Assay