Glun2b

Manufactured by Cell Signaling Technology

Sourced in United States

GluN2B is a subunit of the N-methyl-D-aspartate (NMDA) receptor, a type of ionotropic glutamate receptor. The GluN2B subunit plays a critical role in the function of the NMDA receptor, which is involved in synaptic transmission and plasticity in the central nervous system.

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Lab products found in correlation

Glun2a, by Cell Signaling Technology (4 mentions) Gapdh, by Cell Signaling Technology (3 mentions) Glua1, by Cell Signaling Technology (2 mentions) Glua1, by Abcam (2 mentions) C fos, by Cell Signaling Technology (1 mentions) Anti actin antibody, by BD (1 mentions) Anti mouse or anti rabbit igg hrp linked secondary antibodies, by Cell Signaling Technology (1 mentions) Rabbit anti trkb, by Santa Cruz Biotechnology (1 mentions) Rabbit anti bdnf, by Santa Cruz Biotechnology (1 mentions) Mouse anti β actin, by Merck Group (1 mentions) P camkii, by Cell Signaling Technology (1 mentions) Camkii, by Cell Signaling Technology (1 mentions) Polyvinylidene fluoride pvdf membrane, by Merck Group (1 mentions) Esketamine, by Jiangsu Hengrui Medicine (1 mentions) Protease inhibitor, by Thermo Fisher Scientific (1 mentions) Psd 95, by Merck Group (1 mentions) Image lab 6, by Bio-Rad (1 mentions) Pierce 660 nm assay, by Thermo Fisher Scientific (1 mentions) Non fat dry milk, by Bio-Rad (1 mentions) β actin monoclonal, by Abcam (1 mentions)

9 protocols using glun2b

Antibody Preparation for Neuroscience Research

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H89 and antibodies directed against NeuN and C/EBPβ were purchased from EMD Millipore. 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. Anti-actin antibody was from BD Transduction Laboratories. The anti-GluN1 (clone N308/48) and anti-GluA1 (N355/1) monoclonal antibodies were obtained from the UC Davis/NIH NeuroMab Facility.

Vingtdeux V., Chang E.H., Frattini S.A., Zhao H., Chandakkar P., Adrien L., Strohl J.J., Gibson E.L., Ohmoto M., Matsumoto I., Huerta P.T, & Marambaud P. (2016). CALHM1 deficiency impairs cerebral neuron activity and memory flexibility in mice. Scientific Reports, 6, 24250.

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Hippocampal Protein Expression Analysis

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Mice were killed by cervical dislocation at 16 weeks of age and their brains were collected and stored at −80°C. The hippocampus was bilaterally dissected and separated into dorsal and ventral hippocampus (~50/50). Protein extraction and Western blot analysis were performed as previously described (Hill et al., 2014 (link)). Primary antibodies were rabbit anti-BDNF (1:200, Santa Cruz Biotechnology Inc, Santa Cruz, CA, USA or Almone Labs, Israel), rabbit anti-NT-4 (1:200, Santa Cruz), rabbit anti-pTrkB Y705 (1:1,000, Signalway Antibody LLC, Maryland, USA), rabbit anti-pTrkB Y515 (1:1,000, Abcam, Cambridge, MA, USA), rabbit anti-pTrkB Y816 (1:500, Millipore, CA, USA), rabbit anti-TrkB (1:1,000, Santa Cruz), rabbit anti-NMDAR subunit 1 (GluNR1, 1:1,000, Cell Signaling Technology Inc, Danvers, MA, USA), rabbit NMDAR subunit 2A (GluN2A, 1:1,000, Cell Signaling Technology), rabbit NMDAR subunit 2B (GluN2B, 1:1,000, Cell Signaling Technology), or mouse anti-β-actin (1:10,000, Sigma-Aldrich). Secondary antibodies included anti-mouse or anti-rabbit IgG HRP-linked secondary antibodies (1: 2,000; Cell Signaling Technology; Danvers, MA, USA).

Grech A.M., Ratnayake U., Hannan A.J., van den Buuse M, & Hill R.A. (2018). Sex-Dependent Effects of Environmental Enrichment on Spatial Memory and Brain-Derived Neurotrophic Factor (BDNF) Signaling in a Developmental “Two-Hit” Mouse Model Combining BDNF Haploinsufficiency and Chronic Glucocorticoid Stimulation. Frontiers in Behavioral Neuroscience, 12, 227.

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Pharmacological Neuromodulation Assay

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Remifentanil was purchased from the Yichang Renfu Pharmaceutical Industry. Esketamine was bought from Jiangsu Hengrui Medicine. N-Methyl-d-aspartic acid (NMDA) was purchased from Selleckchem. Polyvinylidene fluoride (PVDF) membrane was bought from Millipore. Antibodies used in this study were purchased from Abcam (CaMKII and p-CaMKII) and Cell Signaling Technology (GluN2B and GAPDH). All other chemicals were purchased from Sigma-Aldrich.

Wang J., Feng Y., Qi Z., Li J., Chen Z., Zhang J, & Zhu D. (2024). The role and mechanism of esketamine in preventing and treating remifentanil-induced hyperalgesia based on the NMDA receptor–CaMKII pathway. Open Life Sciences, 19(1), 20220816.

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Western Blot Analysis of Synaptic Proteins

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Neurons were collected in RIPA buffer with protease inhibitors (1 : 100, Fisher; Cat #: 78444), sonicated, and centrifuged (20 min, 14 000 g, 4°C). Protein in supernatant was quantified (Pierce 660 nm assay, Thermo Scientific, Waltham, MA, USA; Cat #: PI22663). Thirty‐five micrograms of protein were run on a 4–20% gradient polyacrylamide gel and transferred onto a 0.22 µm nitrocellulose membrane. Blots were blocked in 5% non‐fat dry milk (Bio‐Rad, Hercules, CA, USA, Cat #: 1706404) and incubated with primary antibody as follows: GluN2A (1 : 1000, Cell Signaling, Beverly, MA, USA, overnight, 4°C, RRID: AB_2112295), GluN2B (1 : 1000, Cell Signaling, overnight, 4°C, RRID: AB‐1264223), PSD‐95 (1 : 5000, Millipore, Burlington, MA, USA, overnight, 4°C, RRID: AB_10807979) or β‐actin HRP (1 : 5000, Cell Signaling, 1 h, (20℃), RRID: AB_1903890), followed by host‐specific secondary antibody for 1 h at 20℃ (1 : 2000 of goat anti‐rabbit HRP (Cell Signaling, RRID: AB_2099233) or goat anti‐mouse Alexa Fluor^® 647 (Probes, RRID: AB_2535804). Blots were imaged (Bio‐Rad Chemidoc) and band intensity was analyzed using Image Lab 6.0.1 (Bio‐Rad).

Bowers M.S., Cacheaux L.P., Sahu S.U., Schmidt M.E., Sennello J.A., Leaderbrand K., Khan M.A., Kroes R.A, & Moskal J.R. (2019). NYX‐2925 induces metabotropic N‐methyl‐d‐aspartate receptor (NMDAR) signaling that enhances synaptic NMDAR and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor. Journal of Neurochemistry, 152(5), 523-541.

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Apoer2 and Glutamate Receptor Antibodies

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Apoer2 antibodies: tail-specific (C-terminal antibody, rabbit polyclonal affinity purified, 1:500), and extracellular antibody (against the ligand binding domain, rabbit polyclonal serum, 1:1000). Commercial antibodies: Tuj-1 monoclonal (1:10,000, Millipore), β-actin monoclonal (1:2,000, Abcam), HA-specific polyclonal for detection of ADAM10 expression (1:2000, Sigma). Glutamate receptor antibodies: GluA1 (1:1000, rabbit polyclonal, Abcam), GluA2/3 (1:2000, rabbit polyclonal, Abcam), GluN2A (1:500, Cell Signaling), GluN2B (1:1000, Cell Signaling).

Wasser C.R., Masiulis I., Durakoglugil M.S., Lane-Donovan C., Xian X., Beffert U., Agarwala A., Hammer R.E, & Herz J. (2014). Differential splicing and glycosylation of Apoer2 alters synaptic plasticity and fear learning. Science signaling, 7(353), ra113.

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Fractionation and Western Blot Analysis of Mouse Brain Regions

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Cortex, prefrontal cortex, hippocampus, cerebellum, striatum and hippocampal PSD fractions were prepared from three pairs of 8-week-old Ctrl and cKO male mouse brains as previously described61 (link). Adult neural progenitors were isolated from adult mouse hippocampi (C57BL/6) and cultured as previously described62 (link)63 (link). Protein lysate subjected to western blot analysis were separated on SDS–polyacrylamide gel electrophoresis and probed with specific antibodies; GluN1 (rabbit, 1:1,000; Epitomics, 2824-1,), GluN2A (Cell Signaling Technology, 4205, 1:1,000), GluN2B (Cell Signaling Technology, 4212, 1:1,000), GluA1 (rabbit, 1:1,000; Epitomics, 3861-1), GluA2 (rabbit, 1:1,000; Epitomics, 3520-1), CRMP2 (rabbit, 1:1,000; Cell Signaling Technology, 9393 and Sigma,), p-CRMP2 (rabbit, 1:1,000; Epitomics, 5799-1), SYP (mouse, 1:1,000; Santa Cruz, Sc-17750), PSD-95 (rabbit, 1:1,000; Abcam, ab18258), Actin (mouse, 1:1,000; Cell Signaling Technology, 3700), GAPDH (rabbit, 1:1,000; Cell Signaling Technology, 2118). The relative amount of β-Actin or GAPDH was used as loading control. For quantification, the densitometry measurement of each band (Image J) was first normalized to that of Actin or GAPDH and then averaged from at least three independent experiments. Images have been cropped for presentation. Full size images are presented in Supplementary Fig. 4.

Zhang H., Kang E., Wang Y., Yang C., Yu H., Wang Q., Chen Z., Zhang C., Christian K.M., Song H., Ming G.L, & Xu Z. (2016). Brain-specific Crmp2 deletion leads to neuronal development deficits and behavioural impairments in mice. Nature Communications, 7, 11773.

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Hippocampal Western Blot Analysis

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After 4 weeks of feeding, mice were sacrificed at 2 timepoints (ZT 10 and 22) via cervical dislocation with the aid of night vision goggles for ZT 22. Brains were harvested and whole hippocampus was extracted intact, placed in sucrose homogenization buffer, and snap frozen in liquid nitrogen. Samples were homogenized, prepared with equivalent protein concentrations, and loaded on Invitrogen 4-12% Bis-tris gels using 15μg of homogenate per lane. Blots were probed for pERK1/2 (1:1000, Cell Signaling, #9102), ERK (1:1000, BD Bioscience, #610124), GluN2B (1:500, Cell Signaling, #14544), GluA1 (1:500, Cell Signaling, #13185) and GAPDH (1:5000, Cell Signaling, #2118). Three inter-blot control samples were run on every blot and used for normalization when comparing across blots. Within each blot, all bands were first normalized to the average inter-blot control and then a loading control (GAPDH or ERK). Blots were imaged and analyzed using Li-Cor Image Studio Lite version 5.2.

Davis J.A., Paul J.R., McMeekin L.J., Nason S.R., Antipenko J.P., Yates S.D., Cowell R.M., Habegger K.M, & Gamble K.L. (2020). High fat and high sucrose diets impair time-of-day differences in spatial working memory of male mice. Obesity (Silver Spring, Md.), 28(12), 2347-2356.

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Western Blot Analysis of Epigenetic and Synaptic Proteins

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Tissue samples were made into protein samples by RIPA lysis buffer and prepared for protein electrophoresis. After electrophoresis, the gels were transferred to polyvinylidene fluoride (PVDF) membranes, then incubated with the indicated antibodies with gentle shaking overnight at 4 °C. The antibodies used were HDAC1 (1:1,000, Cell Signaling Technology, #34589), HDAC2 (1:1,000, Cell Signaling Technology, #57156), HDAC3 (1:1,000, Cell Signaling Technology, #85057), HDAC4 (1:1,000, Cell Signaling Technology, #15164), HDAC5 (1:1,000, Cell Signaling Technology, #20458), HDAC6 (1:1,000, Cell Signaling Technology, #7558), GLT-1 (1:2,000, Cell Signaling Technology, #3838), GLAST (1:2,000, Cell Signaling Technology, #5684), GluN2B (1:1,000, Cell Signaling Technology, #14544), p-GluN2B (1:1,000, Cell Signaling Technology, #5355), GluA1 (1:1,000, Cell Signaling Technology, #13185), p-GluA1 (1:1,000, Cell Signaling Technology, 75574), and β-actin (1:2,000, Cell Signaling Technology, #4967). Next, the PVDF membranes were incubated with specific HRP-conjugated secondary antibodies, and the protein bands were visualized using enhanced chemiluminescence (ECL, Bio-Rad, California, USA). ImageJ software (National Institutes of Health, USA) was employed to quantify the band densities.

Tao K., Cai Q., Zhang X., Zhu L., Liu Z., Li F., Wang Q., Liu L, & Feng D. (2020). Astrocytic histone deacetylase 2 facilitates delayed depression and memory impairment after subarachnoid hemorrhage by negatively regulating glutamate transporter-1. Annals of Translational Medicine, 8(11), 691.

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Protein Expression Analysis in Motor Cortex

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Total proteins were extracted from the stimulated motor cortex of all groups sacri ced 24 h after the last stimulation by using ice cold RIPA buffer. The protein concentrations were assayed by using a BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Protein lysates (40 µg) were loaded onto 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinyldi uoride (PVDF) membranes. The membranes were blocked for 2 h by using 5% non-fat dry milk blocking buffer. Then, the blots were incubated overnight at 4°C with primary antibodies against GAPDH, BDNF, CREB, pCREB Ser133 , PSD-95, GluA1, pGluA1 Ser831 , GluN2B, pGluN2B Tyr1070 , PI3K, pPI3K, AKT, pAKT, mTOR, mTOR (1:1000, Cell Signaling Technology, MA, USA). After washing thrice for 5 min in phosphate-buffered saline and 0.1% Tween 20 (PBST), membranes were incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA). After washing thrice, the proteins were visualized with automatic chemiluminescence apparatus (Tanon Biotechnology, Shanghai, China) and quanti ed by densitometry.

Liu X., Qi S., Hou L., Liu Y, & Wang X. (2024). Noninvasive Deep Brain Stimulation via Temporal Interference Electric Fields Enhanced Motor Performance of Mice and Its Neuroplasticity Mechanisms. Molecular neurobiology, 61(6).

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