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Phospho p70s6k1 thr389

Manufactured by Cell Signaling Technology
Sourced in United States

Phospho-p70S6K1 (Thr389) is a lab equipment product that detects the phosphorylation of the p70 S6 kinase 1 (p70S6K1) protein at the threonine 389 residue. This phosphorylation event is a key regulatory step in the activation of the p70S6K1 protein, which plays a critical role in cell growth and proliferation.

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4 protocols using phospho p70s6k1 thr389

1

Immunoblotting Analysis of Cellular Signaling

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AS160 (#07-741), Flag (#F7425), α-tubulin (#T6074), and phospho-TBC1D1 Ser237 (#07-2268) antibodies were purchased from MerckMilliporeSigma. ACC (#3676), phospho-ACC1 Ser79 (#3661), Akt (#4691), phospho-Akt Ser473 (#4060), phospho-Akt Thr308 (#9275) AMPKα (#2532), phospho-AMPKα Thr172 (#2535), phospho-AS160 Thr649 (#8881), ERK1/2 (#4695), phospho-ERK1/2 Thr202/Tyr204 (#4370), GSK3β (#9315), phospho-GSK3β Ser9 (#9322), HSP90 (#4874), p70S6K1 (#2708), phospho-p70S6K1 Thr389 (#9234), Raptor (#2280), phospho-Raptor Ser792 (#2083), TBC1D1 (#4629), ULK1 (#8054), phospho-ULK1 Ser555 (#5869), and vinculin (#13901) antibodies were purchased from Cell Signaling Technology.
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2

MALDI-TOF Mass Spectrometry Proteomics

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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).
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3

Investigating Chansu Injection Effects

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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.
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4

Ovarian Cancer Cell Line Characterization

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The mTOR inhibitor, rapamycin, was purchased from Calbiochem (San Diego, CA, USA). Propidium iodide (PI) was from Sigma. Anti-AE2 from Affinity (Cincinnati, OH, USA).The antibodies against p16, CDK4 and Cyclin D1 were from Santa Cruz Biotechnology (Santa Cruz, CA), and the antibodies against p-AKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p70S6K1, phospho-p70S6K1 (Thr389) and GAPDH were from Cell Signaling Technology (Beverly, MA). The horseradish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG were from Beyotime. The human ovarian cancer cell lines OVCAR3, SK-OV-3, HO-8910, COC1 and A2780 (American Type Culture Collection; Rockville, MD, USA) were maintained in RPMI 1640 (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone) and penicillin/streptomycin, and cultured at 37 °C in a 5% CO2 incubator.
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