The cytoplasmic/nuclear protein extraction kit (Beyotime Biotechnology, Wuhan, China) was used to isolate cytoplasmic/nucleus protein. In brief, first, the cells were pelleted, washed and then dissolved in reagent A. After 5 s vortex, the tubes were incubated for 12 min on ice. Next, reagent B was added and the tubes were vortex for 5 s again and then incubated on ice. 1 min later, the samples were immediately centrifuged for 5 min at 14,000×g at 4°C and the supernatant was transferred into another new tube and frozen for further analysis (Cytoplasmic fraction). Then, the remaining supernatant was decanted and the pellet was resuspended in reagent C (nuclear protein extraction). After another vortex for 30 min, the tubes were centrifuged for 10 min at 14,000×g, and the supernatant was transferred to a new tube for further analysis (Nuclear fraction). Finally, the cytoplasmic/nuclear fractions were used to perform Western blot for the detection of NF-κB (p65) level.
Nuclear cytoplasmic protein extraction kit
Manufactured by Beyotime
Sourced in China
The Nuclear/Cytoplasmic Protein Extraction Kit is a tool designed to efficiently separate and extract nuclear and cytoplasmic proteins from cells. It provides a reliable method for the isolation and purification of these distinct protein fractions for further analysis and study.
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Lab products found in correlation
Ripa buffer, by Beyotime (5 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Abin1, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Westernbright ecl, by Advansta (1 mentions)
Enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Loading buffer, by Beyotime (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Mitochondria cytosol fractionation kit, by Beyotime (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Solarbio (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Anti survivin, by Abcam (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti cyclind, by Cell Signaling Technology (1 mentions)
Mouse antibody against β actin, by Merck Group (1 mentions)
Anti histone 3, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
11 protocols using nuclear cytoplasmic protein extraction kit
1
Nuclear and Cytoplasmic Protein Extraction
2
Western Blot Analysis of Inflammation Proteins
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Proteins were extracted from the peri-infarct cortex using RIPA buffer (Beyotime, China). A cytoplasmic/nuclear protein extraction kit (Beyotime) was used to extract cytoplasmic and nuclear proteins. Western blot was performed as described previously [39 (link)]. Briefly, proteins were separated on 10% gels and transferred to PVDF membranes. After blocking with 5% skim milk, membranes were incubated with the primary antibodies at 4°C overnight. The following primary antibodies against specific proteins were used: ABIN1 (#4664, Cell Signaling Technology, USA, 1 : 1000), A20 (#5630, Cell Signaling Technology, USA, 1 : 1000), NF-κB p65 (#8242, Cell Signaling Technology, 1 : 1000), IκBα (18220-1-AP, Proteintech, USA, 1 : 000), phospho-IκBα (Ser32) (#2859, Cell Signaling Technology, 1 : 1000), GAPDH (10494-1-AP, Proteintech, USA, 1 : 1000), and histone H3 (#3638, Cell Signaling Technology, 1 : 1000). Membranes were washed with TBST and immersed in the secondary antibodies at 37°C for 1 h. After washes with TBST, immunoreactive bands were detected using WesternBright ECL (Advansta, USA). Membranes were scanned and analyzed using a Fusion FX5 analysis system (Vilber Lourmat Fusion FX 7 Spectra, France).
3
Nuclear Protein Extraction and NF-κB Binding Assay
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Nuclear proteins were prepared from BMDMs using a cytoplasmic/nuclear protein extraction kit (Beyotime, Shanghai, China). Nuclear proteins were incubated with biotin-labeled oligonucleotide probes containing NF-κB binding site in a binding buffer containing poly(dI-dC), MgCl2, NaCl, EDTA, DTT, and glycerol. The mixture was loaded onto a 4% nondenaturing polyacrylamide gel. After electrophoresis, the products were transferred onto a Hybond-N+ membrane and visualized by using the enhanced chemiluminescence reagent and an Omega Lum G imaging system (Aplegen, Pleasanton, CA).
4
NF-κB p65 Expression in HPV-Infected Tissues
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Western immunoblot analysis was used to detect the expression of NF-κB p65 in nuclei isolated from HPV-infected and normal tissues. Dermal and subcutaneous tissues were removed from the specimens and the epidermis was sliced into small sections to prepare the epithelial nuclear proteins using a cytoplasmic/nuclear protein extraction kit (Beyotime Co., Shanghai, China). Equal quantities of cytoplasmic and nuclear extracts were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked overnight at 4°C in phosphate-buffered saline and 0.1% Tween-20 buffer containing 5% non-fat dried milk proteins. The membranes were then blotted for 2 h at room temperature with the primary anti-NF-κB p65 antibody diluted at 1:500. The membrane-bound protein-antibody complexes were labeled with horseradish peroxidase-conjugated anti-IgG diluted at 1:3,000. Histone H3 was used as an equal protein loading control. An enhanced chemiluminescence system (Pierce Biotechnology, Inc., Rockford, IL, USA) was used for detection.
5
Cytoplasmic and Nuclear Protein Extraction
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Cell cytoplasmic/nuclear fraction for protein isolation was performed using a Cytoplasmic & Nuclear Protein Extraction Kit (P0027, Beyotime) according to the manufacturer’s protocol. Briefly, cells were lysed in cytosolic extraction buffer on ice for 15 min, vortexed for 5 s, and centrifuged at 12,000 rpm for 5 min. The supernatant was collected to obtain the cytosolic fraction. The pellet was then lysed in nuclear extraction buffer on ice for 30 min with vortexing every 2 min and then centrifuged at 16,000 rpm for 10 min. The supernatant was collected to obtain the nuclear fraction. Loading buffer (P0015L, Beyotime) was added to each cytoplasmic or nuclear fraction sample, and the samples were boiled for 10 min at 100 °C for SDS–PAGE and western blot analysis.
6
Protein Extraction and Western Blot Analysis
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After treatment with different conditions as described in the figure legends, cells were harvested and lysed on ice in RIPA buffer (Beyotime, Haimen, China). After centrifuged at 12 000 g for 15 min, the clear supernatant was collected and used as the cell protein extract. For preparation of cytosolic and mitochondrial fractions, cells were collected and lysed in lysis buffer using mitochondria/cytosol fractionation kit (Beyotime, Haimen, China). Cytosol and mitochondrial extracts were isolated according to the manufacturer's protocol. Nuclear protein was extracted according to the manufacturer's instructions of the nuclear/cytoplasmic protein extraction kit (Beyotime, Haimen, China). Protein concentration was determined using the bicinchoninic acid protein assay kit (Beyotime, Haimen, China). Equivalent amounts of protein (30 μg) from each sample were subjected to electrophoresis on a SDS-polyacrylamide gel, and then transferred on to PVDF membranes (Millipore, Bedford, MA, USA). Target antigens were detected with primary antibodies and subsequently secondary antibodies. Immunoreactive bands were then developed using an ECL chemiluminescence reagent (Pierce, Rockford, IL, USA), according to the manufacturer's instruction. The density of each band was measured using the public domain NIH Image software (open source Image J software available at http://rsb.info.nih.gov/ij/ ).
7
Protein Extraction and Western Blotting
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Total protein from cells and tissues was extracted using RIPA lysis buffer (Beyotime). Cytoplasmic and nuclear proteins were extracted by sequentially disrupting the cell membrane and nuclear membrane through an osmotic pressure gradient using the Nuclear/Cytoplasmic Protein Extraction Kit (Beyotime). The protein extraction solutions were quantified using the BCA kit (Solarbio) and then thermally denatured using a metal bath. Proteins of different mass sizes were separated by vertical SDS-PAGE and then transferred to PVDF membranes. The PVDF membranes were incubated in blocking solution for 1 hour and then incubated with primary antibodies, followed by chemiluminescent detection using the ECL method. The specific primary antibody information is listed in Supplementary Information Table S1 .
8
Quantification of Ubiquitinated NRF2 in Kidney Cells
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Kidney tissues and HK2 and NRK-49 F cells were lysed with RIPA buffer (P0013B, Beyotime, Shanghai, China) containing a protease inhibitor cocktail (11,697,498,001, Roche, Indianapolis, USA). Nuclear and cytoplasmic fractions were isolated from fresh renal cortex tissue with a Nuclear/Cytoplasmic Protein Extraction Kit (P0028, Beyotime, Shanghai, China). Equal amounts of each sample were acquired, and WB was conducted according to conventional instructions. The primary and secondary antibodies are shown in the supplementary Table 2 . The bound antibodies were visualized with an enhanced chemiluminescence detection system (Bio-Rad, CA, USA). Subsequently, the abundance of proteins was evaluated using ImageJ (NIH, USA). The co-IP assay was conducted with protein A/G beads (MedChemExpress, HY-K0202) as indicated by the instructions. In brief, cells were lysed in co-IP lysis buffer. The supernatant and NRF2 antibodies were incubated together and then centrifuged with protein A/G. After washing and eluting the sample, WB was performed to detect ubiquitin-linked NRF2.
9
Protein Extraction and Western Blot Analysis
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Total protein was extracted from cells with RIPA buffer (Beyotime, China). Nuclear protein and cytoplasmic protein were extracted with a nuclear cytoplasmic protein extraction kit (Beyotime, China). All protein concentrations were measured by BCA protein assay kit (Beyotime, China). Approximately 40 μg protein extract per sample was separated using a SDS polyacrylamide gel and transferred onto the PVDF membrane (Millipore, USA), and 5% bovine serum albumin was used to block membrane. The membranes were incubated with rabbit anti-GFP (1:1000, Beyotime, China), anti-β-catenin (1:1000, Cell Signaling, USA), anti-Survivin (1:1000, Abcam, USA), anti-cyclin D (1:1000, Cell Signaling, USA), anti-c-myc (1:1000, Abcam, USA), anti-β-catenin Ser45 phosphorylation (1:1000, Cell Signaling, USA), and mouse antibody against β-actin (1:10,000, Sigma, USA), anti-Histone 3 (1:1000, Sigma, USA) overnight at 4°C, followed by incubation for 1hr with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:10,000). After extensive washing in TBST, the expression levels of the protein were detected by Quantity-one software (Bio-Rad Laboratories, USA) using the ECL kit.
10
Protein Extraction and Western Blot Analysis
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Cell nuclear and cytoplasmic proteins were extracted using a nuclear-cytoplasmic protein extraction kit (#P0028, Beyotime), following the manufacturer’s instructions. Cell and tissue proteins were extracted using RIPA buffer (#P0013B, Beyotime). Protein concentrations were measured with the enhanced BCA Protein Assay Kit (#P0012, Beyotime). SDS-PAGEs of the samples (30-50 μg) were conducted with 10–15% polyacrylamide gel. The electrophoresed proteins were electro-transferred into nitrocellulose filter membranes that were blocked with Tris-buffered saline, containing 5% non-fat milk. The membranes were incubated with primary antibodies overnight at 4°C and incubated with secondary antibodies at the room temperature for 1 h. The Odyssey Infrared Imaging System (Licor Biosciences) was used to visualize the bands, and Image J software was used to analyze the relative intensities of the protein bands. The antibodies used: anti-YAP (#14074, Cell Signaling Technology), anti-pYAP (#4911, Cell Signaling Technology), anti-AMPK (#5831, Cell Signaling Technology), anti-pAMPK (#bs-4002R, Bioss), anti-CCND (#55506, Cell Signaling Technology), anti-LKB1 (#3050, Cell Signaling Technology), anti-CDK4 (#bs-0633R, Bioss), and anti-CCNB (#bs-0572R, Bioss). Antibody information is presented in Supplemental Table S2 .
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