Phospho p70s6k1 thr389

All chemicals and solvents used were purchased from Sigma–Aldrich (St. Louis, MO). Solvents were HPLC grade and were used without further purification. Alpha-cyano-4-hydroxycinnamic acid (CHCA) was used as the MALDI matrix. WCX magnetic beads and Standard Preparation (Peptide Calibration Standard #206195 and Protein Calibration Standard I #206355) were purchased from Bruker Daltonik GmbH (Bremen, Germany).
The anti-α-tubulin antibody was from Epitomic Inc (Burlingame, California). Antibodies directed against 4E-BP1, phospho-4E-BP1 (Ser65), p70S6K1 and phospho-p70S6K1 (Thr389) were from Cell Signaling Technology, Inc (Danvers, USA). An IRDye 680-conjugated goat anti-rabbit IgG secondary antibody was from Rockland Immunochemical Inc (Gilbertsville, PA). The anti-eRF3b antibody directed against the human eRF3b was produced by Sangon (Shanghai, China).

Dulbecco's Modified Eagle Medium (DMEM) and fetal bovine serum (FBS) were purchased, respectively, from Mediatech (Herndon, VA, USA) and Gibco (Logan, UT, USA). Trypsin was from Invitrogen (Grand Island, NY, USA). Type I insulin-like growth factor (IGF-I) (PeproTech, NJ, USA) was rehydrated in 0.1 M acetic acid for preparing a stock solution (10 ng/ml), aliquoted, and stored at -80°C. The following antibodies were used: mTOR, phospho-mTOR (Ser2448), S6K1, phospho-p70 S6K1 (Thr389), 4E-BP1, Cyclin A, CDK2, Cyclin B1, CDK4, Cyclin D1, Cyclin E, p-Rb, and β-tubulin (Cell Signaling, Beverly, MA, USA).
Chansu injection, provided by Jiangsu Pujin pharmaceutical Co., Ltd., contains major compounds including nine bufadienolides which accounts for more than 90%. And per 1ml Chansu injection contains 96μg bufadienolides. So in this paper, the Chansu injection was represented by the bufadienolides.