Cytoplasmic and nuclear fractions were isolated with NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific) according to manufacture instructions. Briefly, astrocytes cultured in 100mm dishes were detached with phosphate buffered saline (PBS) plus protease inhibitor cocktail (Sigma), and cytoplasmic and nuclear fractions separated. Fractions were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and analyzed by western blot. Primary antibodies for rabbit anti-Nrf2 (1:1000) and rabbit anti-Fyn (1:1000) were purchased from Cell Signaling Technology (CST) (Danvers, MA). Mouse anti-α tubulin (CST) and rabbit anti-histone H3 (abcam) were used as cytosolic and nuclear loading controls, respectively. Secondary antibodies were goat anti-rabbit HRP and goat anti-mouse HRP (Thermo Scientific).
Rabbit anti nrf2
Manufactured by Cell Signaling Technology
Sourced in United States
Rabbit anti-Nrf2 is a primary antibody that specifically recognizes the Nrf2 (Nuclear Factor Erythroid 2-Related Factor 2) protein. Nrf2 is a transcription factor that plays a crucial role in the regulation of antioxidant response elements and the expression of genes involved in the cellular defense against oxidative stress.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (2 mentions)
Rabbit anti fak, by Cell Signaling Technology (2 mentions)
Goat anti rabbit hrp, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse hrp, by Thermo Fisher Scientific (1 mentions)
Mouse anti α tubulin, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Rabbit anti fyn, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Rabbit anti histone h3, by Abcam (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
Rabbit anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Gel doc, by Bio-Rad (1 mentions)
Enhanced chemiluminescence system, by GE Healthcare (1 mentions)
Mouse anti lamin b, by Abcam (1 mentions)
Rabbit anti ho 1, by OriGene (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Sds polyacrylamide gel electrophoresis, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
9 protocols using rabbit anti nrf2
1
Cytoplasmic and Nuclear Protein Fractionation
2
Western Blot Analysis of Nrf2, Bach1, and HO-1
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Proteins from total cell lysates or nuclear fractions were denatured in Laemmli buffer and then subjected to SDS-polyacrylamide gel electrophoresis (200 Volt for 50 min, Mini Protean precast TGX Gel - percentage of acrylamide is specified in supplementary information - Bio-Rad, Milan, Italy), followed by electroblotting (100 Volt for 50 min) on PVDF membrane (GE Healthcare, Amersham Place, UK). Immunodetection was performed using rabbit anti Nrf2 (1:2000, Cell Signaling), mouse anti Bach1 (1:1000, Santa Cruz Biothec) and rabbit anti HO-1 (1:2000, Origene). After incubation with specific secondary antibodies (GE Healthcare), the bands were detected by means of an enhanced chemiluminescence system (GE Healthcare). The membranes were stripped using Re-blot plus solution (Chemicon International, CA, USA) and re-probed with rabbit anti GAPDH (loading control for total lysates or cytosolic marker, 1:10000, Santa Cruz Biotech) or mouse anti lamin B (loading control for nuclear proteins, 1:1000, AbCam). Developed films were analysed using a specific software (GelDoc; Bio-Rad).
3
Nrf2 Expression Analysis in Hippocampus
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Dissected hippocampi were immediately immersed individually in a lysis buffer and sonicated for 10 s (n = 3 mice per group). Sodium dodecyl sulfate (SDS) sample buffer (4×) was added to each homogenized sample, and the samples were heated at 100 °C for 10 min. Immunoblotting was performed as described previously [55 (link)]. Briefly, the resolved proteins were separated with SDS-polyacrylamide gel electrophoresis (Bio-Rad, Hercules, CA, USA) and transferred onto membrane by using Trans-Blot Turbo Transfer System (Bio-Rad). The membrane was incubated with rabbit anti-Nrf2 (1:1000; Cell Signaling, Danvers, MA, USA) overnight at 4 °C. After extensive washing and incubation with secondary antibodies for 2 h at room temperature, signals were developed using a chemiluminescence kit (SuperSignal® West Pico PLUS; Thermo Fisher Scientific) and read on a ChemiDoc MP Imaging System (Bio-Rad). To quantify Nrf2 expression, the membrane was re-probed with an antibody for β-actin (1:10,000; Sigma-Aldrich). Several exposure times were used to obtain signals within the linear range, and the bands were quantified using the ImageJ software (NIH, Bethesda, MD, USA).
4
Immunofluorescence Staining of Neural Markers
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The following antibodies were used in this study: chicken anti-MAP2 (catalog number CPCA-MAP2, EnCor Biotechnology, Gainesville, FL, USA); rabbit anti-TH (catalog number AB152, Millipore, Billerica, MA, USA); rabbit anti-Nrf2 (catalog number D1Z9C, Cell Signaling Technology, Danvers, MA, USA); mouse anti-GFAP (catalog number 3670, Cell Signaling Technology); and anti-rabbit IgG-Alexa Fluor 488, anti-mouse IgG-Alexa Fluor 647, and anti-chicken IgG-Alexa Fluor 594 (Invitrogen, Carlsbad, CA, USA).
5
Molecular Signaling Assay Protocol
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Whole cells were lysed with RIPA lysis buffer (Beyotime, China, Cat No.P0013B). After protein quantification with the BCA Protein Assay Kit (Thermo Fisher Scientific, CA, Cat No.23225), equal amounts of proteins were separated on SDS-PAGE, and transferred to a PVDF membrane. Antibodies against the following proteins were used: Rabbit-anti-NRF2, rabbit-anti-MLC2, rabbit-anti-phospho-MLC2 (Thr18/Ser19), rabbit-anti-FAK, rabbit-anti-phospho-FAK (Tyr397), and rabbit-anti-ERR1, all purchased from Cell Signaling (Cat No. 12721, 3672, 3674, 3285, 8556 and 13826, respectively). Mouse-anti-RhoA was purchased from Cytoskeleton (Cat No. ARH03, CA), and mouse-anti-actin was purchased from ProteinTech (Cat No. 60008-1-Ig, CA).
6
Immunofluorescence Analysis of Vascular Smooth Muscle Cells
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VSMCs were seeded into a 24well plate at a density of 3 × 104 cells/ml and cultured for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.2% Triton X-100 for 10 min at RT. After washing, cells were blocked with 2% BSA in PBS for 1 h, removed from the blocking solution, and incubated overnight at 4 °C with rabbit anti-Nrf2 (1:50) (Cell Signaling, Beverly, MA, US), rabbit anti-AhR (1:100) (BioWord, MN, US), rabbit-anti-FAK (1:100) (Cell Signaling, Beverly, MA, US), mouse anti-paxillin (1:300) (Sigma, MO, US), mouse anti-integrin β1 (1:100) (Sigma, MO, US) and actin stains with DyLight™ 594 Phalloidin (1:20) (Cell Signaling, Beverly, MA, US). Then, the cells were gently washed under the cover slip three times with PBS and mounted in fluoroshield™ mounting medium with DAPI (Cell Signaling, Beverly, MA, US). Cells were visualized under a laser scanning confocal microscope (Fluoview FV1000 confocal system, Olympus). To ensure blinded data analysis, immunofluorescence staining were performed by one person, and all images and samples were recorded by another person.
7
Protein Extraction and Western Blot Analysis
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Total proteins from frozen liver tissue specimens and/or HSC-T6 cells were extracted with RIPA lysis buffer (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The Bradford assay was used to determine the protein concentration of samples. Then, equal amounts (8 µg) of each protein sample was separated on 10% SDS-PAGE, transferred onto PVDF membranes that were then blocked with 5% skimmed milk at room temperature for 1 h. After blocking, the membranes were incubated overnight at 4˚C with primary antibodies, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:5,000; cat. no. 7074; Cell Signaling Technology, Inc.) at 37˚C for 45 min. Primary antibodies included rabbit anti-GAPDH (1:1,000; cat. no. 5174; Cell Signaling Technology, Inc.), rabbit anti-Nrf2 (1:3,000; cat. no. ab137550), rabbit anti-HO-1 (1:2,000; cat. no. ab13243) both from Abcam, rabbit anti-p-Smad2C (1:1,000; cat. no. 18338) and rabbit anti-p-Smad2C (1:1,000; cat. no. 3104) both from Cell Signaling Technology, Inc. Then, the proteins bands were visualized with the ECL chemiluminescence system (GE Healthcare). The density of each protein blot was compared with that of GAPDH using ImageJ software (version 1.46r; National Institutes of Health) and was shown as a ratio to the endogenous control. These experiments were repeated three times.
8
Quantification of NF-κB and Nrf2 Liver Cells
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Immunohistochemistry was performed to analyze NF-κB and Nrf2-positive cells in liver tissues. Briefly, liver tissue slides, 5 μm thick, were incubated with primary antibodies rabbit anti-NF-κB (Invitrogen 44-711G) and rabbit anti-Nrf2 (Cell Signaling12721S) diluted 1 : 100 in PBS for 12 h at 4°C; as a secondary antibody, we used a goat anti-rabbit IgG (SIGMA A0545) diluted 1 : 200 in PBS. The peroxidase activity was developed with Sigma Fast Diaminobenzidine (Sigma-Aldrich D12384); the number of NF-κB and Nrf2-positive cells was counted in slides and reported as cells/mm2. Data was documented in a Zeiss Axioscope 40/40FL microscope (Zeiss, Oberkochen, GE) and analyzed with the Image Pro Plus Software 4.5.1 (Media Cybernetics, Bethesda, MD, USA).
9
Immunofluorescent Visualization of Nrf2
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The cells were plated onto poly-l -lysine–coated slides, fixed in an acetone:methanol solution (1:1) for 10 minutes at −20 °C, and washed twice with PBS for 5 minutes. The cells were then permeabilized in 0.2% Triton X-100 for 5 minutes and washed twice with PBS for 5 minutes. The cells were blocked in 1.5% horse serum in PBS for 30 minutes at room temperature and then incubated overnight in a humidified chamber at 4°C with primary antibodies including rabbit anti-Nrf2 (1:300, Cell Signaling Technology, Danvers, MA). After washing twice with PBS, the cells were incubated with rabbit-FITC secondary antibody (1:500, Sigma-Aldrich, St Louis, MO) for 1 hour at room temperature. They were also stained with 4,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Slides were then washed twice with PBS and mounted with DAKO fluorescence mounting medium (DAKO, Santa Clara, CA) before viewing with Olympus BX51 Fluorescence microscope with MetaMorph software (FISH).
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