| Project/Area Number |
11557015
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 展開研究 |
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| Research Field |
Pathological medical chemistry
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| Research Institution | The University of Tokyo (2000-2001)
Japanese Foundation For Cancer Research (1999) |
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Principal Investigator |
MIYAZONO Kohei The University of Tokyo, Graduate School of Medicine, Professor, 大学院・医学系研究科, 教授 (90209908)
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| Co-Investigator(Kenkyū-buntansha) |
IMAMURA Takeshi The Cancer Institute of the Japanese Foundation for Cancer Research, Associate member, 癌研究所生化学部, 主任研究員 (70264421)
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| Project Period (FY) |
1999 – 2001
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| Project Status |
Completed (Fiscal Year 2001)
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| Budget Amount *help |
¥12,700,000 (Direct Cost: ¥12,700,000)
Fiscal Year 2001: ¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 2000: ¥4,700,000 (Direct Cost: ¥4,700,000)
Fiscal Year 1999: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Keywords | TGF-beta / signal transduction / protein-protein interaction / mass-spectrometry / Smad / DNAP / 骨形成因子 / 構造決定 / 質量分析 / アミノ酸配列 |
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| Research Abstract |
In order to study signaling mechanisms using protein-protein interaction, we have isolated Smad interacting proteins using mass-spectrometry analysis. We transfected Flag-tagged Smad constructs into mammalian cells. Proteins were then immunoprecipitated by Flag antibody followed by separation by SDS-PAGE and silver staining. Proteins were excised and digested by trypsin, and analyzed by mass-spectrometry. We have found that Rab family proteins interact with Smad1 in vitro.
We next tried to isolate Smad3-interacting proteins using DNA affinity purification (DNAP), since Smad3 specifically binds to the CAGA motif. We found that Smad3 binds to CAGA motif only when cells were treated by TGF-beta. Moreover, we found that Smad2 and transcriptional regulators, p300 and c-Ski, also co-precipitate with Smad3 in this assay.
We have found that determination of the concentrations of poly did-C), washing conditions of beads, and incubation periods with DNA are important factors for DNAP. We also found that Dynabeads dramatically reduce background in silver staining, and are useful for this assay. In conclusion, we found that DNAP is a useful method for isolation of proteins, which bind to DNA.
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