Proteins of mice hippocampus and frontal cortex was prepared after 3 injections of SCE, AA, SCE and AA mixture (10 mg/kg) using an RIPA buffer consisting of 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholate and 1% Nonidet P-40. A 10% phosphatase inhibitor and 10% protease inhibitor cocktail (Roche, Basel, Switzerland) was added before using the RIPA buffer. Next, 12 μg of protein was mixed with a 5X sample buffer and loaded on SDS-PAGE gel. The Protein sample was run on gel by electrophoresis and transferred by 200 mA to polyvinylidene fluoride (PVDF) membrane (Millipore, MA, USA). The membrane was blocked by 5% skim milk and incubated with primary antibodies including anti-PSD95 (Thermo Scientific, MA, USA), GluR1 (Abcam, Cambridge, UK), GAD65 (Abcam, Cambridge, UK), Gephyrin (Synaptic Systems, Goettingen, Germany), and α-Tubulin (Sigma-Aldrich, MO, USA) antibodies at 4 °C overnight. The membrane was incubated with a secondary anti-mouse or rabbit IgG horseradish peroxidase antibody (HRP, Pierce Biotechnology, MA, USA), which corresponds to the primary antibody host, for 1 h at room temperature and each protein band was visualized by the ECL system (Thermo Scientific, MA, USA). For ECL detection, we used medical X-ray film blue (AGFA CP-BU NEW, Belgium), developer solution, and fixer solution for the ECL detection.
Glur1
Manufactured by Abcam
Sourced in United States
GluR1 is a protein that functions as a subunit of the AMPA-type glutamate receptor, which is involved in excitatory neurotransmission in the central nervous system. It plays a role in synaptic plasticity and is important for learning and memory processes.
Automatically generated - may contain errors
Lab products found in correlation
Glur2, by Abcam (3 mentions)
Lsm 710 confocal microscope, by Zeiss (3 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Gad65, by Abcam (1 mentions)
Ecl system, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Anti psd95, by Thermo Fisher Scientific (1 mentions)
Gephyrin, by Synaptic Systems (1 mentions)
Vglut1, by Abcam (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Tuj 1 antibody, by Fortrea (1 mentions)
Vglut2, by Merck Group (1 mentions)
Homer 1, by R&D Systems (1 mentions)
Syn 1, by Merck Group (1 mentions)
Tau 5, by Thermo Fisher Scientific (1 mentions)
Mab377, by Cell Signaling Technology (1 mentions)
P gsk3β, by Cell Signaling Technology (1 mentions)
Na934, by Cytiva (1 mentions)
Gsk3β, by BD (1 mentions)
8 protocols using glur1
1
Hippocampus and Frontal Cortex Protein Profiling
2
Antibody Procurement for Protein Analysis
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Antibodies were purchased from the following companies: anti-β-actin from Sigma (St. Louis, MO, USA), vGluT1, GluR1, GluR2 antibodies from Abcam (Cambridgeshire, England), GAD, NR1, NR2A, NR2B antibodies from Millipore (Billerica, MA, USA), and Tuj-1 antibody from Covance (Princeton, NJ, USA).
3
Immunofluorescent Mapping of Synaptic Markers
4
Detailed Immunodetection Procedures
5
Anesthesia and Protein Analysis in Mice
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The ketamine (100 mg/kg)/xylazine (10 mg/kg, i. p.) were used to anesthetize mice. After the mice were decapitated, the hippocampus were rapidly removed and homogenized in a lysis buffer (in mM: Tris-HCl 50, NaCl 150, EDTA 5, NaF 10, sodium orthovanadate 1, phenylmethylsulfonyl fluoride 1, 1% Triton X-100, 0.5% sodium deoxycholate and protease inhibitor cocktail). After that, total proteins (20 μg) were separated by SDS-PAGE and blotted onto a polyvinilidene fluoride (PVDF) membrane. The membranes were incubated in antibodies of phospho-ERK1/2 (1:1,000; CST, USA); phospho-GluR1, phospho-GluR2, phospho-GluN2A and phospho-GluN2B (1:1,000; Abcam, United Kingdom); phospho-Src (1:1,000; Millipore, USA). After visualization, the membranes were treated with stripping buffer (Restore; Pierce) for 15 min, and then incubated with antibodies of ERK1/2 (1:1,000; CST, USA); GluR1, GluR2, GluN2A and GluN2B (1:1,000; Abcam, United Kingdom); Src (1:1,000; Millipore, USA) or β-actin (1:1,000; CST, USA). Ras activation assay was implemented according to the instructions of the company (Cell biolabs). The small GTPase is tested by Western blot using a target-specific antibody included in the kit.
6
Protein Expression Analysis in Cells
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The cells were harvested and lysed in RIPA Lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing 1% protease inhibitor. Equal weights of total protein were separated by SDS/PAGE. After the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA), the blots were incubated with blocking buffer (1 X TBST and 5% skim milk) for 1 h at room temperature and then hybridized with the primary antibodies GluR1 (1:1000; Abcam, Cambridge, UK), GluR2 (1:1000; Abcam, Cambridge, MA, USA), PICK1 (1:1000; Cell Signaling Technology, Denver, MA, USA), PKCα (1:1000; Abcam, Cambridge, MA, USA), PINK1 (1:1000; Cell Signaling Technology), Parkin (1:1000; Abcam, Cambridge, MA, USA) and Homer 1b/1c (1:700; Synaptic Systems, Goettingen, Germany) overnight at 4 °C, followed by incubation with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature. The blots were obtained by X-ray film exposure.
7
Hippocampal Synaptosomal Western Blotting
8
Investigating CREB1-NMDAR1/GLUR1 Interactions
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Immunoprecipitation was used to survey the binding status between CREB1 and NMDAR1 or GLUR1. Approximately 100 mg of hippocampaus tissues was homogenised and added RIPA lysis buffer (Beyotime, Nantong, China). Equal amounts of the proteins were incubated with 2 µl of Rabbit IgG (1:100, Abcam, Cambridge, UK) as polyclonal–Isotype control or 10 µl of CREB1 (1:20, Santa Cruz, Dallas, USA) or 4 µl of GLUR1 (1:50, Abcam, Cambridge, UK) or 4 µl of NMDAR1 (1:50, Cell Signal, Boston, USA) antibody 8 h at 4°C followed by incubation of 20 µl of Protein A + G Agarose beads (Beyotime, Nantong, China) overnight at 4°C. The mixture was centrifuged (3000 rpm, 5 min, 4°C) and rinsed hree times with RIPA lysis buffer. The mixture was boiled with 1 × Western blot loading buffer for 5 min. After spinning (3000 rpm, 5 min, 4°C), the supernatants were subjected to Western blot with same set of antibodies as above (Ref. 37 ).
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