—33 × 106 LN229 shB7-H3 and LN229 control cells were plated in 6 well plates for 6 h. Afterward, they were co-incubated with 106 freshly isolated NK cells for 45 min. NK-cells were carefully mobilized with 5 ml of ice-cold phosphate buffered saline (PBS) and pelleted (500 g, 5 min, 4°C). After resuspension in freeze and thaw buffer (PBS, 1 mM MgCl2, 2 mM Na-orthovanadate, Phosphatase-Inhibitor cocktail 1 and 3 [Sigma-Aldrich]) cells were treated with 2 freezes (liquid-nitrogen) and thaw cycles. Afterward, cells were pelleted (13.000 rpm, 4°C, 1 h) to separate the cytoplasmic lysis fraction (supernatant) from the membrane fraction. The latter one was lysed for 30 min in digitonin lysis buffer (150 mM NaCl, 1 mM MgCl2, 10 mMTris-HCl [pH8], 1% digitonin, 2 mM Na-orthovanadate, Phosphatase-Inhibitor cocktail 1 and 3 [Sigma-Aldrich]) and afterward pelleted (13.000 rpm, 4°C, 10 min). Supernatant was kept for analysis of phosphoproteome by mass spectrometry (detailed in Supplemental Methods ).
Phosphatase inhibitor cocktail 1 and 3
Manufactured by Merck Group
The Phosphatase-Inhibitor cocktail 1 and 3 are solutions designed to inhibit the activity of phosphatases. Phosphatases are enzymes that remove phosphate groups from proteins, affecting various cellular processes. These cocktails can be used to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using phosphatase inhibitor cocktail 1 and 3
1
Quantitative Analysis of NK Cell-Induced Phosphorylation
2
Preparation and Analysis of Myocyte Cell Lysates
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The preparation of cell lysates from cultivated myocytes has previously been described (20 (link)). For GST pull-down assays, differentiated myotubes were washed briefly in ice-cold PBS and homogenized in 20 mm Tris–HCl/pH 8.0, 1 mm EDTA, 200 mm NaCl and 0.5% NP40, supplemented with complete mini protease inhibitor cocktail (Roche) and phosphatase inhibitor cocktail 1 and 3 (Sigma-Aldrich). Extracts were centrifuged at 14 000g for 10 min at 4°C, and supernatants were collected and used for GST pull-down assays. Beads coupled with GST-tagged recombinant proteins were incubated (rotation) with cell lysates for 2 h at 4°C. After washing three times with 20 mm Tris–HCl/pH 7.4, 0.1 mm EDTA, 300 mm NaCl, 0.5% Nonidet P-40 and 1% Triton X-100, the beads were heated in 5× SDS sample buffer for 5 min at 95°C, and the supernatants subjected to SDS-PAGE and IB. Bait and prey proteins were detected using anti-GST and anti-plectin antibodies, respectively.
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