For nuclear extract purification, cells were resuspended in hypotonic solution (10 mM Hepes [pH 7.9], 10 mM KCl, 1.5 mM MgCl2, 0.5 mM DTT, protease inhibitors [Roche], and phosphatase inhibitors [Sigma-Aldrich]), incubated on ice for 15 min and centrifuged at 13,000 rpm for 20 s. The supernatant containing the cytoplasmic fraction was stored and the pellet was resuspended in nuclear extraction buffer containing 20 mM Hepes [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 0.5 mM DTT, 25% glycerol, protease inhibitors (Roche), and phosphatase inhibitors (Sigma-Aldrich), incubated for 30 min at 4°C and centrifuged at 13,000 rpm for 5 min. NF-κB–specific transcription factor DNA-binding activity in nuclear extracts was performed using the NF-κB (mouse p65) transcription factor assay kit from Cayman Chemical Co., according to the manufacturer’s instructions.
Phosphatase inhibitor
Manufactured by Merck Group
Sourced in United States, Germany, Switzerland, China, Italy, France, Macao, United Kingdom, Sao Tome and Principe
Phosphatase inhibitors are a class of chemical compounds used in laboratory research to inhibit the activity of phosphatase enzymes. Phosphatases are responsible for the removal of phosphate groups from various molecules, and their inhibition can be useful in studying signal transduction pathways and other biological processes involving phosphorylation. These inhibitors are commonly used in cell and molecular biology experiments to preserve the phosphorylation state of proteins during sample preparation and analysis.
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Lab products found in correlation
Protease inhibitor, by Merck Group (251 mentions)
Protease inhibitor, by Roche (113 mentions)
Protease inhibitor cocktail, by Merck Group (74 mentions)
Protease inhibitor cocktail, by Roche (63 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (61 mentions)
Ripa buffer, by Merck Group (56 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (47 mentions)
Ripa buffer, by Thermo Fisher Scientific (44 mentions)
Pvdf membrane, by Merck Group (34 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (22 mentions)
β actin, by Merck Group (21 mentions)
Pvdf membrane, by Bio-Rad (21 mentions)
Nitrocellulose membrane, by Bio-Rad (20 mentions)
Polyvinylidene difluoride membrane, by Merck Group (20 mentions)
Bca assay, by Thermo Fisher Scientific (19 mentions)
Luminata classico, by Merck Group (16 mentions)
Anti β actin, by Merck Group (16 mentions)
Ripa lysis buffer, by Merck Group (16 mentions)
Proteinase inhibitor, by Merck Group (15 mentions)
Stripping solution, by Bio-Rad (15 mentions)
852 protocols using phosphatase inhibitor
1
Nuclear Extract Purification and NF-κB Assay
2
Mammalian Nuclei Isolation and Nuclear Extract Preparation
3
MAPK/SAPK Signaling Pathway Profiling
4
Hippocampal Protein Extraction and Western Blotting
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Mice were euthanized and had their hippocampus dissected and flash frozen. Flash-frozen tissue was homogenized in a T-PER tissue protein extraction reagent, and supplemented with protease (Roche Applied Science, IN, USA) and phosphatase inhibitors (Millipore, MA, USA). The homogenized tissues were centrifuged at 4 °C for 30 min. The supernatant (soluble fraction) was stored at − 80 °C. Western blotting was performed as previously described [49 (link)]. Protein extract was denatured and run in precast Novex gels (Life Technologies). Proteins were then transferred to nitrocellulose membranes using an iBlot (Life Technologies). Membranes were then blocked in 5% powdered milk in Tris-buffered saline with Tween (TBST). Primary antibodies were incubated in 5% milk in TBST overnight. Membranes were washed in TBST the following day and then incubated in fluorescent secondary antibody. The membranes were then washed again, and imaged and quantified using a LI-COR Odyssey CLx (LI-COR Biosciences) and Image Studio (version 1.0.11, LI-COR Biosciences).
5
Dehydrocrenatidine Cytotoxicity Evaluation in Liver Cancer Cells
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Huh-7 and Sk-hep-1 cells were seeded onto 6 cm dishes with 5 × 105 cells per dish, and subsequently treated with 0, 5, 10 and 20 μM of dehydrocrenatidine at 37 °C for 24 h. Next, proteins were extracted from the cells using RIPA buffer containing a mixture of protease inhibitors and phosphatase inhibitors (Millipore Sigma, St. Louis, MO, USA). The proteins were subjected to SDS-PAGE electrophoresis and subsequently transferred onto 0.22 mm polyvinylidene difluoride membranes (PVDF, Millipore Sigma). The membranes were blocked with 5% non-fat milk for 1 h and incubated with indicated primary antibodies overnight at 4 °C. Afterwards, the corresponding horseradish peroxidase-conjugated secondary antibodies were added to the membranes, followed by detection of the signal using chemiluminescent (ECL). Finally, the membranes were photographed with a chemiluminescence fluorescence Image Quant LAS 4000 (GE Healthcare, Berlin, Germany) biomolecule imaging system.
6
Ephrin-B2 Receptor Activation Assay
7
NGF-Induced Signaling Pathways in PC12 Cells
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For immunoprecipitation experiments, PC12 cells were used when they were at 70–80% confluent. Cells were starved from serum for 4 h and subsequently treated with 100 ng/mL NGF (Alomone Labs, cat# N-100) or 500 nM of each compound for 30 min. Cells were then lysed in Pierce™ IP Lysis Buffer (Thermo Fischer Scientific, cat# 87787) containing proteases (Sigma-Aldrich, cat# 539131) and phosphatase inhibitors (Millipore, cat# 524629). Lysates were then immunoprecipitated overnight at 4°C with TrkA antibody (1:100, Millipore, cat# 06-574) followed by 4 h incubation with protein G-plus agarose beads (Santa Cruz Biotechnology, cat# sc-2002). Beads were then collected, washed three times with lysis buffer, resuspended in SDS loading buffer, and subjected to Western blot against phosphorylated Tyrosine (1:1000, R&D systems, cat# BAM1676). Whole-cell lysates were subjected to Western blot against TrkA (1:1,000, Millipore, cat# 06-574), phosphorylated Akt (1:1,000, Cell Signaling, cat# 9271S), phosphorylated Erk1/2 (1:1,000, Cell Signaling, cat# 9101), total Akt (1:1,000, Cell Signaling, cat# 4,691), and total Erk1/2 (1:1000, Cell Signaling, cat# 9,194).
8
Western Blot Analysis of Protein Lysates
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Cell lysates were prepared in radioimmunoprecipitation assay buffer, supplemented with proteinase inhibitors (1:100, Millipore) and phosphatase inhibitors (1:100, Millipore) and protein concentration determined using the (bicinchoninic acid assay BCA) method (23227, Pierce BCA protein assay kit, Thermo Fisher). Western blotting was performed using 4–12% polyacrylamide gels (or 3–8% tris-acetate gel for detection of 53BP1) with 10 µg of protein, followed by methanol-based wet transfer and chemiluminescence detection (Amersham ECL detection reagent, GE Healthcare). Uncropped gels are shown in Supplementary Figure 8 .
9
Protein Extraction and Western Blot Analysis
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Cultured cells were washed twice with cold phosphate-buffered saline (PBS), harvested in 1 ml of PBS, and lysed in 200 μl of radioimmunoprecipitation assay (RIPA) buffer containing EDTA-free protease (Thermo Fisher Scientific) and phosphatase inhibitors (Millipore). Nuclear extracts were prepared using NE-PER (Thermo Fisher Scientific) following the manufacturer’s instructions. Flash-frozen and pulverized tissues were resuspended in RIPA buffer (50 mg in 500 μl) containing protease and phosphatase inhibitors as before. Protein concentrations were determined using a BCA Protein Assay Kit (Thermo Fisher Scientific), and equal amounts of proteins were subjected to SDS–polyacrylamide gel electrophoresis using 4 to 12% SDS–polyacrylamide gels. Proteins were transferred to nitrocellulose membranes, blocked with casein in tris-buffered saline for 1 hour at room temperature, and incubated with primary antibodies overnight at 4°C, followed by incubation with horseradish peroxidase–linked secondary antibodies for 1 hour at room temperature. Blots were developed using Clarity Western ECL Substrate in a ChemiDoc XRS+ imaging system (Bio-Rad). Band intensities were quantified using ImageLab 5.0 (Bio-Rad) and normalized to the housekeeping protein GAPDH unless otherwise specified.
10
Brain Dissection and Fractionation
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Mice were killed by CO2 asphyxiation and their brains were removed and sagittally bisected. Half of the brain was fixed in 4% paraformaldehyde and used for immunohistochemical experiments. The other half was collected and stored at −80°C until use. Frozen brains (without cerebellum) were processed as described previously (Caccamo et al., 2013 ). Briefly, brains were homogenized in a solution of tissue protein extraction reagent (T‐PER; ThermoFisher Scientific) containing 0.7 mg mL−1 of pepstatin A supplemented with a complete mini protease inhibitor tablet (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (Millipore, Billerica, MA, USA). The homogenized mixtures were centrifuged at 4°C for 1 h at 100 000 g, and the resulting supernatant was stored as the soluble fraction. The pellet was then homogenized in 70% formic acid and centrifuged as described above. The supernatant was stored as the insoluble fraction.
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