Primary fibroblasts were serum-starved for 18 h before treatment with 100 nM insulin for 5 min. Cells were lysed in 1× lysis buffer (20 mM Tris–HCl (pH 7.5), 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton; Cell Signaling) supplemented with 1× COMPLETE protease inhibitors (Roche) and 1× phosphatase inhibitors I and II (Sigma-Aldrich). Cell lysates containing 150 μg of total protein were incubated with 5 μg α-INSR antibody (Cell Signaling, 3025), or 5 μg α-IGF1R antibody (Cell Signaling, 3018) at 4°C overnight. Immunocomplex was pulled down using Protein A-Sepharose (GE Healthcare). Input and immunoprecipitation samples were analyzed by Western blot using α-phosphotyrosine antibody (1:1,000; Millipore, 4G10 Platinum), or the above α-INSR (1:1,000) and α-IGF1R (1:1,000) antibodies. Twenty percent of input lysates were loaded as a control.
Phosphatase inhibitors 1 and 2
Manufactured by Merck Group
Sourced in United States
Phosphatase inhibitors I and II are a set of reagents used in biochemical and cell biology research. These inhibitors are designed to temporarily block the activity of phosphatase enzymes, which play a role in the regulation of various cellular processes. The core function of these inhibitors is to provide a controlled environment for studying the effects of phosphatase activity on specific biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (3 mentions)
Protease inhibitor cocktail, by Cell Signaling Technology (2 mentions)
Agarose gel, by Bio-Rad (2 mentions)
Pierce bca kit, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
4g10 platinum, by Merck Group (1 mentions)
Protein a sepharose, by GE Healthcare (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Ubiquitin, by Cell Signaling Technology (1 mentions)
Sds page gel, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Ero1α, by Santa Cruz Biotechnology (1 mentions)
Eif2α, by Cell Signaling Technology (1 mentions)
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (1 mentions)
P eif2α, by Cell Signaling Technology (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
6 protocols using phosphatase inhibitors 1 and 2
1
Insulin and IGF1 Receptor Activation
2
T cell Ubiquitination and Metabolism
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T cells were lysed in RIPA buffer (Sigma, St. Louis, MO, USA) supplemented with protease inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA) and phosphatase inhibitors I and II (Sigma, St. Louis, MO, USA). Protein concentrations were normalized using Pierce BCA Kit (Thermo Fisher, Waltham, MA, USA) and loaded to 4%–10% agarose gels (BioRad, Hercules, CA, USA). Primary antibodies for Ubiquitin, ATF4, TCF7, HK2, β-actin, Tubulin, and anti-rabbit secondary were obtained from Cell Signaling Technologies, Danvers, MA, USA. p-ACC was obtained from Thermo Fisher (Waltham, MA, USA).
3
Radiation-Induced Apoptosis in Glioma Cells
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Western immunoblots of U87MG hGRM1-positive human glioma cells either treated with riluzole for 24 hours or no treatment were conducted. Both sets of cells were irradiated at 2 or 4 Gy. Lysates were made at 24, 48 or 72 hours after irradiation. Protein lysates were prepared by washing cells with PBS, adding extraction buffer (50 mM Tris, 150 mM NaCl, 1mM EDTA, pH 8.0, 1% NP40, 5% glycerol, 1mM Dithiothreitol, complete protease inhibitor cocktail (Roche) and phosphatase inhibitors I and II (Sigma). The samples were resolved in 10% SDS-PAGE gels (Bio-Rad), after which they were transferred onto nitrocellulose membranes. Membranes were blocked with 5% milk and 1% bovine serum albumin and then probed with antibodies against PARP, cleaved caspase-3, or γH2AX. The same blot was probed with α-tubulin or loading controls to show equal loading.
4
Protein Extraction and Immunoblotting for T-Cell Analysis
5
Serum Deprivation and Inhibitor Effects
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Sub-confluent cells (50%) serum-deprived for 24 hours were treated with either EGF or OA and in the presence or absence of pharmacological inhibitors for the indicated times. When indicated, cells were treated with ribosomal inhibitor Geneticin (G418) [21 (link)] at 1 mg/ml 30 minutes before the main stimulation to prevent de novo synthesis of proteins. Cells were harvested and lysed with 20 mM TRIS pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.2 mM MgCl2, 0.5% Nonidet p40, 1 mM DTT, 25 mM NaF and 25 mM beta-glycerophosphate supplemented with phosphatase inhibitors (I and II) and protease inhibitors (all from Sigma-Aldrich, St Louis, MO, USA). The extracts were analysed by SDS-PAGE followed by Western blot (WB) using the appropriate antibodies. WBs were developed using Horseradish peroxidase substrate (ECL) (GE Healthcare, GE, Little Chalfont, United Kingdom) and images were taken with a Chemidoc XRS® (Bio-Rad, Hercules, Ca, USA).
6
Western Blot Protocol for Protein Analysis
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Cells were scraped in lysis buffer [mPER (Thermo Scientific, #78501) +Protease Inhibitor (Thermo Scientific, #78425) +Phosphatase Inhibitors I and II (Sigma-Aldrich, P2850 and P5726)], spun for 10 min at 4°C, and then supernatants were transferred to new tubes. Protein concentrations were determined with a BCA kit (Thermo Scientific, #23227). Proteins were separated by SDS-PAGE and then transferred to PVDF membranes. Non-specific binding was blocked by incubating membranes with TBST +5% non-fat milk for 30 min. Membranes were then incubated overnight with primary antibodies. After washing with TBST, membranes were incubated with the appropriate HRP-conjugated secondary antibodies. Bound antibodies were detected with SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, #34075).
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