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2 protocols using anti cyclin e

1

Cell Viability and Protein Expression Assay

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Cell culture media, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), hematoxylin and eosin were purchased from Sigma. Foetal bovine serum (FBS) and antibiotics were from Life Technologies and Immobilon P (PVDF) membranes from Millipore. OOS was provided by Catalysis, S.L. (Madrid, Spain). The Cytometric Bead Array Mouse Inflammation kit was purchased to BD biosciences. Other generic chemicals were purchased from Sigma Chemical Co., Roche Biochemicals or Merck.
The origin of the different antibodies used in the Western blotting analyses were as follows: the anti-GAPDH, anti-p21, anti-Wee1, anti-CDC2/CDK1, anti-pCDC2, anti-Cyclin B and anti-Cyclin D1 antibodies were purchased from Santa Cruz Biotechnology; the anti-BUBR1, anti-Rb, anti-Cyclin A, anti-Cyclin D3 and anti-Cyclin E from BD Biosciences; the anti-p27 and the anti-pRbS780 from Cell Signaling technology; the anti-Calnexin from Stressgen and the anti-pHistone H3 from Millipore. The horseradish peroxidase-conjugated secondary antibodies were from Bio-Rad.
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2

Tanshinone Bioactivity Evaluation Protocol

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Bovine serum albumin, sulforhodamine B (SRB), propidium iodide (PI), anti-β-actin antibody, dimethyl sulfoxide, and ribonuclease A (RNase A) were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Dulbecco’s Modified Eagle Medium (DMEM), FBS, and antibiotic-antimycotic solution were purchased from HyClone Laboratories, Inc. (South Logan, UT, USA). Anti-cyclin A, anti-CDK4, anti-CDK2, anti-cMyc, and anti-p21 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-p-Rb (S807/811), anti-Rb, anti-p-Akt (Ser473), anti-Akt, anti-p-mTOR (Ser2448), anti-mTOR, anti-p-p70S6K1 (Thr389), anti-p70S6K1, anti-p-4EBP1 (Thr37/46), anti-4EBP1, anti-p-PDK1 (Ser241), anti-p-AMPK (Thr172), and anti-AMPK were obtained from Cell Signaling (Danvers, MA, USA). Anti-cyclin D1 and anti-Cyclin E were purchased from BD Biosciences (San Jose, CA, USA). Four tanshinones (tanshinone I, tanshinone IIA, cryptotanshinone, and DHTS; Fig. 1) were isolated by Dr. Seung Ho Lee (Yeungnam University, Korea) from the roots of S. miltiorrhiza Bunge (Labiatae) and were provided through the Research Center for Standardization of Herbal Medicines in Korea.
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