Mouse brains were extracted rapidly on ice and then sliced into 1 mm sections, and dorsal hippocampus was removed with a 12 gauge punch and immediately frozen on dry ice. Both human and mouse samples were homogenized by light sonication in modified RIPA buffer (10 mM Tris base, 150 mM sodium chloride, 1 mM EDTA, 0.1% sodium dodecyl sulfate, 1% Triton X-100, 1% sodium deoxycholate, pH 7.4, protease and phosphatase inhibitors [Sigma Aldrich]). Concentration was measured using DC Protein Assay (BioRad) and gel samples were normalized for total protein. Proteins were separated on 4–15% polyacrylamaide gradient gels (Criterion System, BioRad), and Western blotting was performed using chemiluminescence (SuperSignal West Dura, Thermo Scientific). Total protein was assayed using Swift Membrane Stain (G Biosciences) and proteins were quantified using ImageJ software (NIH). Primary antibodies were used to detect FosB isoforms (5G4; 1:500; Cell signaling, 2251), GluA2/3 (1:1,000; Millipore, 07–598), CaMKIIα (1:1,000; Millipore, 05–532), cdk5 (1:1,000; Santa cruz, sc-173), GAPDH (1:20,000; Cell Signaling, 21185).
Glua2 3
Manufactured by Merck Group
Sourced in United Kingdom
GluA2/3 is a laboratory equipment product by Merck Group. It is used for the detection and quantification of GluA2 and GluA3 subunits of the AMPA-type glutamate receptor in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Glua1, by Merck Group (6 mentions)
Actin, by Merck Group (4 mentions)
Glua4, by Merck Group (3 mentions)
Enhanced chemi luminescence (ecl), by PerkinElmer (2 mentions)
Protease and phosphatase inhibitor, by Merck Group (1 mentions)
Supersignal west dura, by Thermo Fisher Scientific (1 mentions)
Criterion system, by Bio-Rad (1 mentions)
Swift membrane stain, by G Biosciences (1 mentions)
Camkiiα, by Merck Group (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Pglua1 s845, by Merck Group (1 mentions)
Synaptophysin, by Merck Group (1 mentions)
Tubulin, by Merck Group (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Gephyrin, by Synaptic Systems (1 mentions)
Gephyrin, by BD (1 mentions)
Affi gel columns, by Bio-Rad (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
8 protocols using glua2 3
1
Protein Extraction and Analysis from Mouse Brains
2
Synaptosomal Fractionation and Immunoblotting
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Synaptosomal fractions from DIV14 primary cortical neurons were prepared as described previously [60] (link),[61] (link). Surface biotinylation was performed according to the previous study [60] (link). Equal amounts of protein were loaded on 10% SDS-PAGE gel and transferred to the nitrocellulose membrane. Membranes were blotted with GluA1 (Millipore, 1∶5,000), GluA2/3 (Millipore, 1∶500), pGluA1(S845) (Millipore, 1∶1,000), calcineurin (Millipore, 1∶1,000 or Santa Cruz Biotechnology, 1∶500), actin (Sigma, 1∶5,000), pCREB (Cell Signaling, 1∶500), and CREB (Cell Signaling, 1∶1,000) antibodies and developed with ECL (Perkin Elmer). Synaptosomes were isolated from at least three independent cultures, and immunoblots were least duplicated for quantitative analysis.
3
Surface Expression of GluA Subunits
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Alterations in the surface expression of GluA subunits were measured using Bis(sulfosuccinimidyl)suberate (BS3) cross-linking methods as described previously (Anderson et al., 2008). Briefly, the nucleus accumbens shell and core were dissected and incubated in 2mM BS3 in artificial cerebrospinal fluid (aCSF) for 30 min at 4°C. The reaction was quenched with 100mM glycine, washed with aCSF, resuspended in lysis buffer (10 mm Tris-HCl, pH 7.4, 5 mm NaF, 1 mm Na3VO4, 1 mm EDTA, 1 mm EGTA) and prepared for immunoblots. The samples were used for western blots as described above using the following antibodies GluA1, GluA2/3, and GluA4 (1:700, Millipore). All bands were normalized to GAPDH.
4
Antibody Dilutions for Western Blot and Immunostaining
5
Fractionation and Analysis of Hippocampal Proteins
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Hippocampi were isolated in cold PBS, flash frozen in liquid nitrogen, and stored at −80°C. Brain lysates, synaptosomes, and PSD fractions were prepared as described previously (Phillips et al., 2001 (link); Jordan et al., 2004 (link)). An equal amount of protein (10 μg) from each fraction was separated by SDS-PAGE. The following antibodies were diluted in TBST (0.2% Tween-20), with 2% BSA: Lrp4 (1:2500; Neuromab, N207/27, Davis, CA), Synaptophysin (1:20,000; Life Technologies, Grand Island, NY), PSD95 (1:1000; Neuromab, K28/43), GluA1 (1:1000; Millipore, Billerica, MA), GluA2/3 (1:500; Millipore), pan-Shank (1:1000; Santa Cruz Biotechnology, C-20, Dallas, TX), NR1 (1:1000; Neuromab, N308/48), GABAAR γ2 (1:1000; PhosphoSolutions, Aurora, CO), NeuN (1:1000; Millipore), Gephryin (1:1000; SYnaptic Systems, Goettingen, Germany), actin (1:2000; Sigma, AC74, St. Louis, MO), N-cadherin (1:1000; BD Transduction Laboratories, 610920, San Jose, CA), PTPR σ (1:500; Protein Tech Group, Chicago, IL), GFAP (1:1000; Sigma), β-tubulin III (1:6000; SYnaptic SYstems). The following antibodies were gifts from P. Scheiffele: Neuroligin 1 (1:2000), Neuroligin 2 (1:2000), Neuroligin 3A (1:2000), and pan-Neuroligin (1:3000).
6
Surface Expression of GluA Subunits
7
Western Blot Analysis of Neuronal Markers
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Brain tissues were homogenized in the sample lysis buffer (62.5 mM Tris-HCl pH 6.8, 2 % SDS, 0.5 % NP-40, 5 mM EDTA) plus the protease inhibitor cocktail (Roche). Protein samples (15-30 µg/lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Memb-ranes were probed with antibodies directed against phosphotyrosine (1:1000, Millipore, Bedford, MA, USA), ErbB4 (1:1000, Santa Cruz Biotechnology). Alter-natively, immunoblots were probed with antibodies directed against excitatory and inhibitory neuronal markers GluA1 (1:500, Millipore), GluA2/3 (1:1000, Millipore), GluA4 (1:1000, Millipore), GluN1 (1:1000, Millipore), GluN2A (1:500, Millipore), GluN2B (1: 500, Millipore), GluN2C (1:500, Millipore), GluN2D (1:1000, Santa Cruz Biotechnology), GAD65/67 (1:1000, Millipore), parvalbumin (1:3000, Abcam, Cambridge UK), tyrosine hydroxylase (1:1000, Millipore) neuregulin-1 (1:300, RandD Systems, Minneapolis, MN), and β-actin (1:2000, Millipore). Immunoreactivity on membranes was detected by peroxidase-conjugated anti-immunoglobulin antibodies followed by chemiluminescence reaction (ECL kit, GE Healthcare) and film exposure. The intensity of an immunoreactive band, whose size matched the authentic molecular weight, was measured by an image processing software, GENETOOLS (Syngene, Cambridge, UK).
8
Western Blot Analysis of Key Synaptic Proteins
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Whole-brain tissue and PSD fraction from mouse brain were prepared as described previously (Restituito et al. 2011 (link); Kim et al. 2015b (link)). Equal amounts of protein were loaded on 10% SDS-PAGE gel and transferred to the nitrocellulose or PVDF membranes. Membranes were blotted with GluA1 (Millipore, 1:5000), GluA2/3 (Millipore, 1:500), GluA1-pS845 (Millipore, 1:1000), actin (Sigma, 1:5000), DARPP-32-pT32 (Millipore, 1:1000), DARPP-32 (BD biosciences, 1:250), and cGKII (Serulle et al. 2007 (link)) (1:1000) antibodies and developed with ECL (PerkinElmer).
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