Endothelial cells were seeded in 10-cm dishes, followed by stimulation (37°C) with or without 100 µM H2O2 for 48 h. Subsequently, the HepG2 cell (Tianjin Saierbio Biotechnology Co., Ltd., Tianjin, China) lysates were prepared with 1% Tris-Triton cell lysis buffer (Cell Signaling Technology, Inc., Danvers, MA, USA) pre-mixed with 1 mM phenylmethanesulfonyl fluoride on ice for 30 min and centrifuged (4°C) at 12,000 × g for 30 min. The supernatants were incubated (4°C) overnight with 30 µl Dynabeads protein A or protein G (Thermo Fisher Scientific, Inc.) pre-coated with anti-S100A4 (1:250, Abcam) antibodies. The immunocomplexes were subjected to western blot analysis. The normal corresponding immunoglobulin G control was assayed simultaneously.
Tris triton cell lysis buffer
Manufactured by Cell Signaling Technology
Sourced in China
Tris-Triton cell lysis buffer is a solution used to extract and solubilize cellular proteins from cell samples. It contains Tris and Triton X-100 as the primary components.
Automatically generated - may contain errors
2 protocols using tris triton cell lysis buffer
1
S100A4 Protein Interaction Assay
2
Immunoprecipitation of β-catenin and Ubiquitin
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Cell lysates were prepared by incubating cells in 1% Tris-Triton cell lysis buffer (Cell Signaling Technology, America) containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and the protease inhibitor cocktail on ice for 30 min, following which, lysates were centrifuged at 12,000 × g for 10 min. The supernatants were incubated overnight with 30 μL Dynabeads Protein A (Life Technologies, America) precoated with anti-β-catenin (Abcam, Cambridge, UK), or anti-ubiquitin (Abcam, Cambridge, UK) antibodies. The immunocomplexes were analyzed by western blots. A normal IgG (Cell Signaling Technology, America) control was assayed simultaneously.
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