Cell Lysis Buffer (10X)

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	- Cells are washed 3 times with cold PBS and removed from the dish by scraping in cell lysis buffer supplemented with inhibitors of protease and protein phosphatase

Protocol tips
- Cells are washed 3 times with cold PBS and removed from the dish by scraping in cell lysis buffer supplemented with inhibitors of protease and protein phosphatase

Publication protocol

The cells were washed 3 times with cold PBS and removed from the dish by scraping in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with inhibitors of protease and protein phosphatase (Roche Diagnostics, Indianapolis, IN, USA). The lysates were then centrifuged at 17,000 × g at 4°C for 10 min. The protein concentrations of the supernatants were determined using the Bio-Rad Protein Assay Dye (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with bovine serum albumin as a standard. The supernatant was stored at −80°C until use. Samples of 40 µg of supernatant protein per lane were separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto nylon membranes for immunoblottiThe membranes were incubated with one of the primary antibodies (diluted 1:1,000) overnight at 4°C, followed by horseradish peroxidase-conjugated secondary antibodies. The immunoreactive blots were visualized with a SuperSignal West Pico Chemiluminescent Substrate detection system (Thermo Fisher Scientific, Rockford, IL, USA) according to the manufacturer's instructions. β-actin (diluted 1:2,000; cat. no. 3700; Cell Signaling Technology) was used as a loading control. A minimum of 3 blots from independent experiments were scanned on an Epson Perfection 1660 Photo scanner, and bands quantified using ImageJ software.ng with specific antibodies.

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