anti phospho n methyl d aspartate receptors subunit 2b nr2b tyr1472  (Cell Signaling Technology Inc)

Journal: medRxiv

Article Title: Urinary Astrocyte-derived Extracellular Vesicles: A Non-invasive Tool for Capturing Human In Vivo Molecular “Movies” of Brain

doi: 10.1101/2024.01.12.24301104

Figure Lengend Snippet: The x-axis in all panels marks the days since admission (Day 0 = admission day). Main treatment included an initial phase of four intravenous infusions of human γ-globulin (dose 25 g, indicated by 4 vertical dot-dash red lines). Simultaneously, glucocorticoid therapy was administered (represented by the green horizontal line above each subfigure, with the line’s width indicating the equivalent dose of methylprednisolone). Additionally, intrathecal methotrexate administration (dose 10 mg, represented by vertical solid yellow lines) and rituximab therapy (dose 100 mg, indicated by vertical long-dash blue lines) were administered. Clinical symptoms were assessed using two scales: (a) the Glasgow Coma Scale (GCS) and (b) the modified Rankin Scale (mRS), showing gradual improvement following treatment. Given the use of the immunotherapy, we measured the (c) percentage of CD19+ cells in the blood, assessed via flow cytometry, dropped to nearly non-existent levels by Day 10. We also monitored NMDAR-antigen (Ag) titres in the CSF. (d) A decrease in NMDAR-Ag titers in the CSF was observed as the treatment progressed. Given that anti-NMDA encephalitis is characterized by the presence of autoantibodies mainly against the NMDAR GluN1 subunit, leading to NMDARs damage through internalization, shedding, and extracellular release, we also measured (e) The concentrations of GluN1 in CSF. As NMDARs are also present on the surface of astrocytes, we simultaneously measured the (f) concentrations of GluN1 in uADEVs (pg/per 1E+10 particles). As the trajectory of GluN1 in uADEVs was also a dynamic signal, composed of various wave components. Therefore, we conducted wavelet analysis using the Morlet wavelet as mother wavelet to identify and separate the significant components of this dynamic signal. (g) Wavelet analysis results of the log(10) GluN1 trajectory in uADEVs are presented, with colors representing power, black lines indicating the highest peak (ridge) in the respective region, and white areas representing significant components. Two significant components were identified, one with a short period (approximately 6-8 days) located below and another with a period of over 20 (the strongest ridge is at approximately 32 days). These two significant components were then reconstructed as follows: (h) The trajectory of the reconstructed significant short-period component (red line), and (i) the long-period component (approximately 32 days, the blue line). The gray dashed line in (h) and (i) represent the original log(10) trajectory (grey dashed lines).

Article Snippet: N-methyl-d-aspartic acid receptor (NMDAR) subunit zeta-1 (GluN1), the pathogenic molecule in anti-NMDAR encephalitis, was measured in CSF and uADEV samples using the enzyme-linked immunosorbent assay (ELISA) (CUSABIO, Catalog# CSB-EL009911HU).

Techniques: Modification, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Use of Steaming Process to Improve Biochemical Activity of Polygonatum sibiricum Polysaccharides against D-Galactose-Induced Memory Impairment in Mice

doi: 10.3390/ijms231911220

Figure Lengend Snippet: Steamed PSPs ameliorate D-gal-induced hippocampal synaptic deficits. ( A – F ) The upper panels show representative blots of Arc/β-actin, GluA1/β-actin, GluA2/β-actin, GluN2A/β-actin, GluN2B/β-actin, p-GluN2B/GluN2B, and PSD95/β-actin, while the lower panel shows Arc/β-actin, GluA1/β-actin, GluA2/β-actin, GluN2A/β-actin, GluN2B/β-actin, p-GluN2B/GluN2B, and PSD95/β-actin quantification data ( n = 6). ( G ) Representative images of synaptic structure in hippocampal area CA1 (Magnification: ×5000). Red arrows indicate the synapses. ( H ) Number of synapses (12 μm 2 ) ( n = 5). Data are expressed as means ± SEM. * p < 0.05.

Article Snippet: Thereafter, protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis at 75 V for 15 min, followed by 115 V for 75 min before transferring at 300 mA onto a nitrocellulose membrane for 2 h. Membranes were blocked with 5% skim milk (dissolved in PBS containing 0.1% Tween-20) for 2 h at room temperature, washed three times with PBS for 10 min each, then incubated at 4 °C overnight in primary antibody against the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) subunits GluA1 (1:1000, CST) and GluA2 (1:1000, CST), the N-methyl-D-aspartate receptor (NMDAR) subunits GluN2A (1:1000, CST), GluN2B (1:1000, CST), and p-GluN2B (1:1000, CST), nuclear factor-erythroid factor 2-related factor 2 (Nrf2, 1:1000, ZEN BIO), heme oxygenase-1 (HO-1, 1:1000, ZEN BIO), NOD-like receptor protein 3 (NLRP3, 1:1000, CST), apoptosis-associated speck like protein (ASC, 1:1000, ZEN BIO), postsynaptic density protein 95 (PSD95, 1:1000, CST), activity-regulated cytoskeletal protein (Arc, 1:1000, Synaptic Systems), and β-actin (1:1000, CST).

Techniques: