Western blot analysis of PI3K/Akt/mTOR-related proteins

Following treatment of the 4T1 cells with fisetin (0, 20, 40 and 80 µM) for 24 h, proteins were extracted from the cells by lysis in radioimmunoprecipitation assay buffer (Applygen Technologies, Inc., Beijing, China) with protease inhibitor cOmplete tablets (Roche Applied Science, Penzberg, Germany) and PhosSTOP phosphatase inhibitor cocktail tablets (Roche Applied Science). Western blotting was performed as previously described (41,42). The primary antibodies, purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA), were anti-mTOR (cat. no. 2983), anti-phosphorylated (p)-mTOR (cat. no. 5536), anti-Akt (cat. no. 9272), anti-p-Akt (cat. no. 9271), anti-PI3K (cat. no. 4263), anti-p-PI3K (cat. no. 13857), anti-B cell lymphoma (Bcl)-2 associated X protein (Bax; cat. no. 14796), anti-Bcl-extra large (Bcl-xL; cat. no. 2764), anti-P70 (cat. no. 2708), and anti-p-p70 (cat. no. 9234). Reference protein (β-actin) antibody (cat. no. E021020-01) was purchased from Beijing GuanXingYun Sci & Tech Co., Ltd. (Beijing, China). The membrane was incubated with above primary antibody in 5% BSA (Amresco, LLC, Solon, OH, USA) at 1:1,000, and then incubated with the Dylight™ 680- (cat. no. 072-06-15-06) or 800-labeled secondary antibody (cat. no. 072-07-18-06; KPL, Inc., Gaithersburg, MD, USA) at 1:8,000. Odyssey infrared imaging system V3.0 (LICOR Biosciences Company, USA) was used for membrane scanning. The signal intensity of each protein was measured with Image-Pro Plus Version 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).