Protocol detail

Protein Immunoblotting Assay Protocol

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Cell pellets were lysed as described previously (19 (link)). Primary antibodies that we used in this study were rabbit anti-phospho Aurora A (T288), rabbit anti-insulin-like growth factor-I receptor (IGF- IR), rabbit anti-insulin receptor (InsR) β, rabbit anti-phospho-IGF-IR/InsR kinase (Thr202/Tyr204), rabbit anti–extracellular signal-regulated protein kinases (ERKs), rabbit anti–phospho-ERKs, rabbit anti–c-Jun N-terminal kinases (JNKs), rabbit anti–phospho-JNKs, rabbit anti–p38, and rabbit anti–phospho-p38 (all from Cell Signaling Technology, Beverly, MA); and mouse anti-Aurora A (BD Biosciences, San Jose, CA), rabbit anti-cyclin B1 (Santa Cruz Biotechnology, Dallas, TX), and mouse anti–β-actin Ab (diluted at 1: 5000; Sigma-Aldrich, St. Louis, MO). Antibodies were diluted at a ratio of 1:1000, unless noted. Signals were detected with use of an Odyssey IR imaging system (LI-COR, Lincoln, NE).

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Kai K., Kondo K., Wang X., Xie X., Pitner M.K., Reyes M.E., Torres-Adorno A.M., Masuda H., Hortobagyi G.N., Bartholomeusz C., Saya H., Tripathy D., Sen S, & Ueno N.T. (2015). Antitumor Activity of KW-2450 Against Triple-Negative Breast Cancer by Inhibiting Aurora A and B Kinases. Molecular cancer therapeutics, 14(12), 2687-2699.

Publication 2015

rabbit anti-phospho Aurora A (T288), (IGF- IR), rabbit anti–p38 rabbit anti–phospho-p38 rabbit anti-cyclin B1 mouse anti–β-actin Ab Odyssey IR imaging system

Actin Anti b1 Antibodies Aurora a Cell Cyclin Extracellular signal regulated kinases Growth factor receptor Igf ir Insulin Insulin receptor Jun n terminal kinases Kinase Mouse Pellets Protein Rabbit

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Variable analysis

independent variables

Treatments or conditions that the researchers manipulated or applied to the cell pellets

dependent variables

Levels or activities of the following proteins measured in the cell pellets:
- Phospho-Aurora A (T288)
- Insulin-like growth factor-I receptor (IGF-IR)
- Insulin receptor (InsR) β
- Phospho-IGF-IR/InsR kinase (Thr202/Tyr204)
- Extracellular signal-regulated protein kinases (ERKs)
- Phospho-ERKs
- c-Jun N-terminal kinases (JNKs)
- Phospho-JNKs
- p38
- Phospho-p38
- Aurora A
- Cyclin B1
- β-actin

control variables

Positive control: None specified
Negative control: None specified

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