Total protein was extracted from hippocampal neurones or hippocampal tissues using Tissue or Cell Total Protein Extraction Kit (Sangon Biotech). Cell-surface and cytosol protein were extracted from hippocampal neurones using Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, Shanghai, China). The concentration of proteins was assessed using BCA Protein Assay Kit (Sangon Biotech) as the protocol described. Equivalent protein (25 μg) from different samples was separated by 10% SDS-PAGE protein electrophoresis, following by transformation onto PVDF membranes (Merck Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk at room temperature for 2 h, and then incubated with the primary antibodies, caspase-1, GluA1 (surface, S), GluA1 (intracellular, I) and Stargazin (1:1000, Abcam) at 4 °C overnight. After the membranes were washed with TBST for several times, horseradish peroxidase-conjugate second antibody (1:1000, Abcam) was incubated with the membranes. β-actin antibody (1:1000, Abcam) was used as a reference protein for normalization. The gray levels of the protein bands were examined by Image J software.
Glua1
Manufactured by Abcam
Sourced in United Kingdom
GluA1 is a subunit of the AMPA-type glutamate receptor, which is a ligand-gated ion channel involved in fast excitatory neurotransmission in the central nervous system. This product can be used for research purposes to study the structure and function of AMPA receptors.
Automatically generated - may contain errors
Lab products found in correlation
Glun2a, by Merck Group (5 mentions)
Psd95, by Abcam (4 mentions)
Glua2, by Abcam (3 mentions)
Glun2b, by Merck Group (3 mentions)
Glua2, by Merck Group (3 mentions)
Mglur5, by Merck Group (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Glun2a, by Cell Signaling Technology (2 mentions)
Glun2b, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Glun1, by Merck Group (2 mentions)
Neuro 2a cells, by American Type Culture Collection (2 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (2 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Puromycin, by Merck Group (2 mentions)
Glun1, by Thermo Fisher Scientific (2 mentions)
Dock4, by Abcam (2 mentions)
Dock4, by Thermo Fisher Scientific (2 mentions)
Anti rb igg hrp, by Cell Signaling Technology (2 mentions)
Nsc23766, by Selleck Chemicals (2 mentions)
19 protocols using glua1
1
Hippocampal Protein Extraction and Analysis
2
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 .
3
Quantifying Brain Region-Specific Protein Profiles
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Protein samples from different regions of mouse brain, including insular cortex and other areas, were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride filters. The filters were incubated overnight at 4 °C with appropriate antibodies. Secondary antibodies conjugated to horseradish peroxidase were added to the filters and then visualized in ECL solution. The visualization was performed via the ImageQuant LAS 4000 mini Molecular Imaging System (GE Healthcare Life Sciences), and the Image J software (NIH) was used for the analysis of band intensity. Antibodies used were as follows: ASIC1a (1:500; Santa Cruz, sc-13905), β-actin (1:1,000; Chemicon, Cat # MAB1501), GAPDH (1:1,000; KangChen, Cat # KC-5G4), GluA1 (1:1,000; Epitomics, Cat # 3861-1), GluA2 (1:1,000; Epitomics, Cat # 3520-1), GluN1 (1:1,000; R&D Systems, Cat # PPS011B), GluN2A (1:1,000; Millipore, Cat # 07-632), GluN2B (1:1,000; Millipore, Cat # MAB5220), PSD-95 (1:1,000; Epitomics, Cat # 2366-1), pGSK3β-Ser9/pGSK3α-Ser21 (1:1,000; Cell Signaling Technology, Cat # 8566) and GSK3β (1:1,000; Cell Signaling Technology, Cat # 12456).
4
Immunofluorescence Staining of Brain Sections
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Brain sections were washed with PBS for three times and were blocked with PBS-containing 3% goat serum (Vector Laboratories, #) and 0.1% Triton X-100 for 1 h at room temperature. Sections were incubated with primary antibodies overnight at 4°C and were washed with PBS. The following primary antibodies were used: GFAP (), mouse anti-Neuronal Nuclei (NeuN, Millipore, MAB377), DCX (), and GluA1 (Abcam, ab1232). On the second day, the samples were taken out and washed with PBS for 3 times, 5 min/time, followed by incubation with the secondary antibodies for 1 h at room temperature. Fluorophore-conjugated secondary antibody was used: goat anti-mouse Alexa Fluor 568 (Invitrogen, A11031), goat anti-rat Alexa Fluor 568 (Invitrogen, A11077), goat anti-rabbit Alexa Fluor 488 (Invitrogen, A11008), and goat anti-mouse Alexa Fluor 488 (Invitrogen, A11001). All the sections were observed and images were taken with a confocal microscope (Leica). The images were analyzed with Imarus software.
5
Hippocampal Protein Expression Analysis
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Four mice per group were anesthetized with intraperitoneal injections of zoletil (90 mg/kg) and xylazine (10 mg/kg), and their hippocampi were collected. The extracted proteins were quantified by the BCA Protein Assay System (Pierce, USA). The proteins were subjected to SDS-PAGE, followed by electro-transfer to PVDF membranes (Millipore, USA). After blocking with 5% skim milk in 0.1% Tris-buffered saline (TBS)/Tween-20, the blots were probed with primary antibodies against GluA1 (1:1000, Abcam), GluN1 (1:1000, Sigma-Aldrich), GluN2A (1:2000, Millipore), NR2B (1:500, BD Transduction Laboratories), and synaptophysin (1:4000, Abcam) overnight at 4 °C. After incubation, the membranes were subsequently incubated for 2 h at room temperature with the appropriate secondary antibodies conjugated with horseradish peroxidase (Vector, USA). The proteins were visualized with an enhanced chemiluminescence kit (Pierce, USA) and band intensities were quantified using the Kodak Gel Logic 2200 imaging system with Molecular Image analysis software (Kodak, USA). β-Actin was used as a loading control (1:10000, Sigma-Aldrich). Data are presented as an average of at least three experiments and analyzed for statistical significance by using Mann-Whitney tests (Prism; Graph Pad, USA).
6
Quantification of Hippocampal Synaptic Proteins
7
Western Blot Analysis of Synaptic Proteins
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The primary antibodies used in Western blot analysis were purchased from the following commercial suppliers: Dock4 (ab85723, 1:1000) and PSD95 (ab2723, 1:1000) were from Abcam; GluA1 (PC246, 1:1000), GluA2 (MAB397, 1:1000), GluN2A (AB1555P, 1:1000), GluN2B (AB1557P and 06-000, 1:1000), Rac1 (05-389, 1:2000), and puromycin (MABE343, 1:10000) were from Merck Millipore; GluN1 (32-0500, 1:1000) was from Invitrogen; Dock4 (WH0009732M1, 1:1000), ɑ-tubulin (T6199, 1:5000), and Flag (F1804, 1:1000) were from Sigma; GAPDH (A01020, 1:5000) was from Abbkine. The secondary antibodies for Western blot were purchased from Cell Signaling Technology (anti-mouse IgG-HRP, 7076s; anti-Rb IgG-HRP, 7074s). Pharmacological inhibitors MG132 and NSC23766 were purchased from Selleck. puromycin was purchased from Merck Millipore. cDNAs of Dock4 and Rac1 and their mutants, and Dock4 shRNA were described previously [27 (link), 32 (link)]. Neuro-2a cells were purchased from ATCC and were routinely tested for mycoplasma contamination-free before use.
8
Hippocampal Protein Expression Analysis
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The hippocampi were homogenized in lysis buffer (RIPA) on ice for 30 min and subsequently centrifuged at 12000 rpm for 5 min at 4 °C. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). Equivalent amounts of proteins were processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot as previously described [29 (link)]. The primary antibodies used in this experiment were Kir4.1 (1:1000, Abcam), AKT (1:1000, CST), Arc (1:1000, CST), PSD95 (1:1000, CST), GluN2A (1:1000, Abcam), GluA1 (1:1000, Abcam), and β-actin (1:1000, CST). Band intensity was densitometrically quantified using Image J software.
9
Immunoblotting of AMPA Receptor Subunits
10
Quantifying Protein Expression in Brain Regions
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The procedure of Western blotting has been previously described (Sun et al., 2009 (link)). Immediately after cocaine prime-induced restatement, rats were rapidly decapitated and brains were removed, flash frozen, and stored in −80°C until used. Brain regions were dissected, including PFC, NAc, caudate-putamen (CPu), bed nucleus of the stria terminalis (BNST), hypothalamus, amygdala (Amy), dorsal hippocampus (dHip), ventral hippocampus, and ventral tegmental area. Protein was extracted by sonication and loaded on 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis gels then transferred to polyvinylidene difluoride membranes. Membranes were then incubated with primary antibody against p-ERK (1:5000), ERK (1:2000) (both from Cell Signaling, Beverly, MA), p-GluA1 Ser845 (1:1000; Millipore, Billerica, MA), GluA1 (1:10000; Abcam, Cambridge, MA), OTR (1:500; Abbiotec, San Diego, CA), and calnexin (1:10000; Enzo Life Sciences, Farmingdale, NY), followed by their appropriate secondary antibodies. Antibody binding was detected by using an enhanced chemiluminescence kit (ECL Plus; GE Healthcare Bio-Sciences, Piscataway, NJ). The integrated density of protein band was measured using ImageJ software (National Institute of Health, Bethesda, MD).
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