Cells transfected with expression vectors were collected and lysed by NETN buffer (25 mm Tris‐HCl pH 8.0, 50 mm NaCl, 0.2 mm EDTA, and 0.1% NP40) with freshly added 1:50 Phosphatase Inhibitor Cocktail 2 (Sigma), 0.1 M DTT (Thermo Fisher Scientific) and sodium fluoride (New England BioLabs). Then 1500 mg of cell lysate was incubated with 3 µg of anti‐CaMK2A (Santa Cruz, CA) antibody with gentle rotation at 4 °C for 4 h. The antibody complex was incubated with pre‐washed protein‐G beads (protein‐G Mag Sepharose Xtra, GE Healthcare) at 4 °C overnight. The complex was washed with NETN buffer five times and eluted. The immunoprecipitants were subjected to western blot analysis conducted as previously described.[5b ]To mimic the hypoxic condition, cells were incubated under low oxygen (1% O2, 5% CO2, and 94% N2) in hypoxia chamber for 24 h; or cells were treated with 100 µm of cobaltous chloride (CoCl2) for 24 h. The primary antibodies included GSTP1 (1:1000; Abcam #ab13849), phospho‐CaMK2A T286 (1:500; Santa Cruz #sc12886‐R), CaMK2A (1:500; Santa Cruz #sc13141), NRF2 (1:1000; Abcam #ab62352), HIF1A (1:1000; Abbexa #abx033664), β‐actin (1:2000; Cell signaling #4970s), E‐cadherin (1:1000; Cell signaling #4065) and VIMENTIN (1:1000; Cell signaling #3932s), TBP (1:2000; Cell signaling #8515s), KEAP1 (1:1000; Santa Cruz #sc365626), and β‐tubulin (1:2000; Invitrogen #PA5‐16863).
Sodium fluoride
Manufactured by New England Biolabs
Sourced in United States
Sodium fluoride is a chemical compound with the formula NaF. It is a white crystalline solid that is commonly used in various laboratory applications. Sodium fluoride serves as a source of fluoride ions, which can be used in various chemical reactions and analytical procedures.
Automatically generated - may contain errors
Lab products found in correlation
Sodium orthovanadate, by New England Biolabs (3 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Anti camk2a, by Santa Cruz Biotechnology (1 mentions)
Protein g mag sepharose xtra, by GE Healthcare (1 mentions)
Pa5 16863, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions)
β tubulin, by Thermo Fisher Scientific (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Phosphatase inhibitor cocktail 2, by Merck Group (1 mentions)
Sorafenib, by LC Laboratories (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Clarity western ecl substrate kit, by Bio-Rad (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Pierce enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Tris gel, by Bio-Rad (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Sds page system, by Bio-Rad (1 mentions)
7 protocols using sodium fluoride
1
Hypoxia-induced CaMK2A Regulation and Signaling
2
Sorafenib Treatment Kinetics in SKHep1 Cells
3
Western Blot Analysis of HEK293A Cells
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HEK293A cells were lysed in NP-40 lysis buffer (containing 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.2% IGEPAL, and 10% glycerol) supplemented with 0.1 M sodium fluoride (NEB, P0759), 10 mM Phenylmethylsulfonyl fluoride (Sigma-Aldrich, PMSF-RO), 10 mM Orthovanadate (NEB, P0758), and Protease inhibitor cocktail (Sigma-Aldrich, P8340-5ML). Protein lysates (30 µg per lane) were resolved on 10% SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% milk in 1x TBS-T buffer (Severn Biotech, 20-7310-10), blotted with primary then secondary antibodies (Supplementary Table 1 ), and developed using a Clarity Western ECL Substrate kit (BioRad, #1705061). Uncropped images of representative blots are available in the Source Data file.
4
Whole Cell Lysate Preparation for Western Blot
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To prepare whole cell lysates of infected cells for Western blot analyses, cells were washed with cold phosphate buffered saline (PBS), then scraped into cold PBS containing protease inhibitors (Roche) plus 5 mM sodium fluoride (New England Biolabs, Ipswich, MA, USA) and 1 mM sodium orthovanadate (New England Biolabs, Ipswich, MA, USA) to inhibit phosphatases. Harvested cells were transferred to a 1.5 mL microfuge tube containing 3× sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer. The lysate was repeatedly passed through a 28 1/2-gauge needle to reduce viscosity, and then heated at 100 °C for 5 min. For Western blot analysis, 10 to 20 μL of whole cell lysate was electrophoresed through SDS 8% polyacrylamide gels. Proteins were transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA), blocked with 3% bovine serum albumin, then probed with appropriate dilutions of primary antibody followed by appropriate dilutions of horseradish peroxidase conjugated secondary antibody. The membranes were treated with Pierce enhanced chemiluminescence substrate (Thermo Scientific, Rockford, IL, USA) and exposed to film.
5
Quantifying Protein Levels via Western Blot
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Protein levels were measured using Western blot analysis [49 (link)]. For whole lung homogenates, the lung tissue was homogenized in RIPA buffer (Thermo Fisher; catalog #89900) and supplemented with protease (Roche; reference #11836170001) and the phosphatase inhibitor sodium orthovanadate (New England Biolabs (NEB), Ipswich, MA, USA; item #P0758) and sodium fluoride (NEB; item #P0759). For cell culture experiments, cellular lysates from were collected in RIPA buffer supplemented with protease and phosphatase inhibitors. Western blotting was performed using an SDS-PAGE system (Bio-Rad, Hercules, CA, USA; item #8658004). Samples were electrophoresed in a 4-20% Tris gel (Bio-Rad; item #4568095) in Tris running buffer, transferred to a PVDF membrane (Bio-Rad; item #1704157), and probed with anti-EGFR (p- and total), anti- ERK1/2 (p- and total), anti-JNK1/2 (p- and total), anti-p38 (p- and total), anti-AKT (p- and total-), anti-HIF1α, anti-PARP (cleaved- and total-), and anti-caspase 3 (cleaved- and total-) primary antibodies (Cell Signaling Technologies, Danvers, MA, USA). Protein band densitometry analysis was completed using ImageJ software.
6
Total Protein Extraction from Cultured Cells
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Total cellular protein was collected from cultured cells following a wash with cold phosphate buffered saline (PBS). Lysis buffer was comprised of RIPA buffer (Sigma-Aldrich) supplemented with 1% protease inhibitor (Sigma-Aldrich), 1% phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich), sodium orthovanadate (2 mM, Sigma-Aldrich), and sodium fluoride (5 mM, New England Biolabs). Lysis buffer was added to adherent cells and incubated on ice for 10 minutes. Alternatively, cells were detached using trypsin, resuspended in growth media, and centrifuged. The cell pellet was resuspended in lysis buffer. Lysates were vortexed 2-3 times over 20 minutes and otherwise kept on ice. Lysates were clarified by centrifugation at 8000 x g for 10 minutes at 4°C. Clarified, soluble protein was transferred to a new tube and stored at -80°C. Protein concentration of these clarified lysates was determined by the Bradford protein quantification assay (Thermo Fisher Scientific).
7
TBC1D4 Phosphorylation Assay
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Recombinant full-length TBC1D4 was purified by IMAC using Ni-NTA resins and used as a substrate in kinase assays. Constitutively active AKT2 was expressed as a GST fusion protein in Sf9 cells and purified as described previously (36 (link)), and purified human AMPK(α1β1γ1) was obtained from Life Technologies. Phosphorylation reactions were carried out at RT for indicated times in the presence of 4 pmol AKT2 or AMPK, 40 mM Tris-HCl pH 7.4, 8 mM MgCl2, 200 μM AMP, 2 mM ATP (or [γ-18O4]ATP), and 0.4 mM dithiothreitol (DTT) in a volume of 200 μl as described (7 (link)), and substrate concentrations are indicated in the figure legends. For dephosphorylation of TBC1D4, the protein was incubated with lambda protein phosphatase (New England Biolabs), 50 mM HEPES, 1 mM MnCl2, 100 mM NaCl, 0.01% Brij 35, and 2 mM DDT, for 30 min at RT. Phosphatase activity was stopped by adding 10 mM sodium orthovanadate and 50 mM sodium fluoride (New England Biolabs).
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