Follow the instructions provided by the manufacturer of the protein extraction kit (Beyotime Biotech Inc.) to extract proteins from hippocampal tissue. The protein was separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio Rad laboratory), and then the protein in the gel was transferred to the polyvinylidene fluoride membrane. Seal the membrane with 5% skim milk for 2 h. Then, the membrane was mixed with GPR30 (1:1000, Abcam) and β‐incubate overnight with actin (1:5000, Abcam). Subsequently, incubate at room temperature with corresponding goat anti mouse and goat anti rabbit (1:5000, Abcam) for 2 h. Finally, the Tanon‐5200 chemiluminescence imaging system was used to detect protein bands, and Image software was used to quantify the gray scale of protein bands.
Gpr30
Manufactured by Abcam
Sourced in United States
GPR30 is a membrane-bound receptor that binds to the hormone estrogen. It is involved in the rapid, non-genomic actions of estrogen and plays a role in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
β actin, by Merck Group (2 mentions)
Goat anti mouse and goat anti rabbit, by Abcam (1 mentions)
Protein extraction kit, by Beyotime (1 mentions)
Actin, by Abcam (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Anti phospho akt ser473, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti cdk2, by Cell Signaling Technology (1 mentions)
Anti n cadherin, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti cyclin a2, by Cell Signaling Technology (1 mentions)
Anti pi3k, by Cell Signaling Technology (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Mmp 9, by Abcam (1 mentions)
Anti gapdh, by Beyotime (1 mentions)
β actin, by Beyotime (1 mentions)
Donkey anti goat igg hrp, by Santa Cruz Biotechnology (1 mentions)
P creb, by Cell Signaling Technology (1 mentions)
Ecl system, by PerkinElmer (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
6 protocols using gpr30
1
Hippocampal Protein Extraction and Analysis
2
Gastric Cancer Cell Lines Protocol
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GC cell lines MGC-803, BGC-823, HGC-27, AGS, MKN-45, and normal gastric epithelial cell GES-1 were obtained from the Cell Resource Center, Peking Union Medical College. All cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Hyclone, Utah, USA) in a humidified atmosphere containing 5%CO2 at 37°C. G1 (Cat# HY-107216), G15 (Cat# HY-103449), and cisplatin (CDDP, Cat# HY-17394) were obtained from MedChemExpress (New Jersey, USA).
The primary antibodies against GPR30 (Cat# ab260033), MMP2 (Cat# ab92536), and MMP9 (Cat# ab76003) were purchased from Abcam (Cambridge, UK). Anti-PI3K (Cat# 4255S), Anti-phospho-AKT (Ser473, Cat# 4060T), Anti-AKT (Cat# 4691T), Anti-CDK2 (Cat# 18048T), Anti-Cyclin A2 (Cat# 4656T), Anti-Cyclin D1 (Cat# 55506T), Anti-N-cadherin (Cat# 13116T), and Anti-Vimentin (Cat# 5741T) antibodies were purchased from Cell Signal Technology (Boston, USA). Anti-GAPDH (Cat# AF1186) and β-actin (Cat# AF5001) antibody were purchased from Beyotime (Shanghai, China).
The primary antibodies against GPR30 (Cat# ab260033), MMP2 (Cat# ab92536), and MMP9 (Cat# ab76003) were purchased from Abcam (Cambridge, UK). Anti-PI3K (Cat# 4255S), Anti-phospho-AKT (Ser473, Cat# 4060T), Anti-AKT (Cat# 4691T), Anti-CDK2 (Cat# 18048T), Anti-Cyclin A2 (Cat# 4656T), Anti-Cyclin D1 (Cat# 55506T), Anti-N-cadherin (Cat# 13116T), and Anti-Vimentin (Cat# 5741T) antibodies were purchased from Cell Signal Technology (Boston, USA). Anti-GAPDH (Cat# AF1186) and β-actin (Cat# AF5001) antibody were purchased from Beyotime (Shanghai, China).
3
Quantitative Western Blot Analysis of Neuronal Markers
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Equal amounts of protein (40 μg) were separated on SDS polyacrylamide gels and electrotransferred onto PVDF membranes (Millipore), which were probed with primary antibodies against β-actin (1:10,000, no. A5316, Sigma-Aldrich), Drebrin (1:2,000, no. 626523, Biorbyt), PJA1 (1:1,000, no. 629950, Biorbyt), Serpina3n (1:2,000, no. AF4709, R&D Systems), CREB (1:1,000, no. 9197, Cell Signaling Technology), p-CREB (1:1,000, no. 9198, Cell Signaling Technology), GPR30 (1:1,000, no. ab39742, Abcam), AACT (1:1,000, no. ab205198, Abcam), and GFAP (1:1,000, no. 3670, Cell Signaling Technology) overnight at 4°C. Subsequently, the membranes were incubated with HRP-conjugated secondary antibodies — including goat anti-mouse IgG-HRP (no. sc-2005, Santa Cruz), goat anti-rabbit IgG-HRP (no. sc-2004, Santa Cruz), and donkey anti-goat IgG-HRP (no. sc-2020, Santa Cruz) — and visualized using an enhanced chemiluminescence (ECL) system (Perkin Elmer). Band density was quantified with ImageJ software. The density of each band was normalized to the β-actin band density. The density ratio of the control group was set as 100%, and the density of the other groups is presented as a percentage of that of the control group.
4
Protein Expression Profiling in Rat PAG
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The segments of PAG were removed rapidly from deeply anesthetized rats, and stored at − 80 °C. The collected tissue samples were homogenized in lysis buffer and centrifuged (4 °C) at 13,000 rpm for 10 min. Protein samples were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10%), and transferred onto a nitrocellulose membrane. The membranes were incubated overnight at 4 °C with primary antibodies against GPR30 (1:500, Abcam), PKA (1:500, R&D Systems), GABAAα4 (1:1000, Santa Cruz Biotechnology), GABAAβ1 (1:1000, Abcam), and GABAAδ (1:1000, Santa Cruz Biotechnology). The membranes were washed and incubated with secondary antibody for 1 h at room temperature. β-actin was used as a loading control. The immune complexes were detected using enhanced chemiluminescence. Density of specific bands was analyzed using Image-Pro Plus software.
5
Molecular Mechanisms of NGR1 in ER Signaling
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NGR1 (molecular weight: 933.15, purity ≥ 98%) was obtained from the Shanghai Winherb Medical S & T Development Co. Ltd (Shanghai, China). ERα, ERβ, and GPR30 receptor agonists and antagonists were procured from Cayman Chemical Inc (Michigan, USA). ERβ, GPR30, and Bax antibodies, as well as secondary antibodies, including goat anti-rabbit immunoglobulin G (IgG) and goat anti-mouse IgG, were purchased from Abcam (Cambridge, England). PI3K, Akt, caspase-3, Bcl-2, ERα, and β-actin antibodies were purchased from Millipore Corporation (CA, USA). Dulbecco's modified Eagle's medium (DMEM) was purchased from Hyclone (Utah, USA). Foetal bovine serum (FBS) was purchased from Gibco (NY, USA). Double antibiotics were purchased from Gibco (NY, USA). The Alexa Fluor 488 Annexin V/propidium iodide (PI) Apoptosis Kit was procured from Life Technologies (CA, USA). The protein extraction and separation reagent were obtained from CW Biotech (Beijing, China). All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
6
Molecular Mechanisms of HMGB1 Signaling
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The following reagents were used: rHMGB1 (Cat No. #1690-HMB-050, R&D Systems, Minneapolis, MN, USA), G1 (Cat. No. 3577/10, TOCRIS, Bioscience, Bristol, UK), G15 (Cat. No. 3678/10, TOCRIS, Bioscience, Bristol, UK). E2, TAM, were obtained from Sigma–Aldrich (St. Louis, MO, USA). U0126, and the LY294002 were purchased from Millipore (Temecula, CA, USA). E2 was dissolved in ethanol and other drugs were solubilized in dimethyl sulfoxide (DMSO; Sigma–Aldrich). The following antibodies were used: anti-HMGB1 neutralizing antibody (Cat No. H00003146-M08, R&D systems, Minneapolis, MN, USA); p-ERK1/2 (Cat No. AP0484P, diluted 1:1000), ERK1/2 (Cat No. BS90472, diluted 1:1000), Akt (Cat No. BS6473, diluted 1:1000) were purchased from Bioworld (St Louis Park, MN, USA). p-Akt (Cat No. 4060, Cell signaling Technology, USA, diluted 1:1000), LC3B (Cat No. 2775 Cell Signaling Technology, diluted 1:1000), P62 (Cat No. 5114, Cell Signaling Technology, diluted 1:1000); Beclin1(Cat No. 210498, diluted 1:1000), GPR30 (Cat No. ab39142, diluted 1:250), HMGB1 (Cat No. ab18256, diluted 1:500), Bcl-2 (Cat No.32124, diluted 1:1000), BAX (Cat No. 182734, diluted 1:1000) were purchased from Abcam (Cambridge, MA, USA), and β-actin (Cat No. TA-09, diluted 1:1000) from Zhongshan Golden Bridge (Beijing, China).
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