TAZ/WWTR1 for diagnosis and treatment of cancer
A cancer, metastatic cancer technology, applied in molecular biology and genetics, cell biology, medicine
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Embodiment 1
[0523] Example 1: Cell Lines and Plasmids
[0524] Cell lines MCF10A, MCF7, MDA-MB-231, Hs578T, ZR-75-1 were purchased from the American Type Culture Collection and maintained in the recommended medium except for MCF10A cells, which were Cultures were performed in DMEM supplemented with 5% horse serum, 20 ng / ml EGF, 0.5 μg / ml hydrocortisone, 100 ng / ml cholera toxin, 10 μg / ml insulin and penicillin / streptomycin. BT-549 cells were cultured in RPMI supplemented with 10% FBS. MDA-MB-435S, T-47D cells were from Lo Ting Ling (Institute of Molecular and Cellular Biology) and maintained in DMEM supplemented with 10% FBS and 10 μg / ml insulin. BT-20, MDA-MB-453 and BT-474 were provided by Yoshiaki Ito (Institute of Molecular and Cellular Biology). BT-20 cells were maintained in MEM supplemented with 10% FBS. MDA-MB-453 cells were maintained in DMEM with 10% FBS supplemented with 10 μg / ml insulin. Amphotropic Phoenix packaging cells were kindly provided by G. Nolan (Stanford Universi...
Embodiment 2
[0526] Example 2: Purification of GST-tagged proteins
[0527] 1 liter of Escherichia coli BL21(DE3) carrying the pGEX-TAZ or pGEX-YAP construct was cultured in LB medium until OD 0.8-1, and was incubated with 0.1 mM IPTG (isopropyl-β-D-thio galactopyranoside) induced overnight. Cells were harvested by centrifugation and lysed by sonication in phosphate buffered saline (PBS). The lysate was spun at 10,000 g for 30 minutes at 40°C and the supernatant was mixed with 0.5 ml Glutathione-Sepharose 4B (Amersham Biosciences) for 2 hours at 4°C. Wash beads four times with PBS. Bound protein was eluted with 5 volumes of 10 mM reduced glutathione in 50 mM Tris, pH 8.
Embodiment 3
[0528] Example 3: Purification of His-tagged proteins
[0529] One liter of E. coli BL21(DE3) carrying the pET-TAZ fusion construct was induced as pGEX-TAZ. By breaking the buffer (100mM Hepes-KOH, pH7.4, 5mM MgCl 2 , 500 mM KCl, 0.1% Triton-X-100, 2 mM β-mercaptoethanol and a protease inhibitor cocktail from Roche Molecular Biochemicals) to lyse the bacteria. The lysates were spun and the supernatant collected and incubated with 1 ml Talon metal affinity resin (Clontech) for 2 hours at 4°C. The resin was washed four times with wash buffer (20 mM Hepes, pH 7.4, 200 mM KCl, 10% glycerol, 10 mM imidazole, and 2 mM β-mercaptoethanol), and washed with 5 volumes of elution buffer (20 mM Hepes, pH 7.4, 200 mM KCl, 10% glycerol, 250 mM imidazole and 2 mM β-mercaptoethanol) elution.
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