Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA.
Application of a protein-blotting procedure to the study of human glucocorticoid receptor interactions with DNA.
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应用蛋白质印迹程序研究人类糖皮质激素受体与 DNA 的相互作用。
DOI：
10.1073/pnas.84.7.1744
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发表时间：
1987
影响因子：
11.1
通讯作者：
Cidlowski,JA
中科院分区：
文献类型：
--
作者：
Silva,CM;Tully,DB;Petch,LA;Jewell,CM;Cidlowski,JA
为了发挥其作用，糖皮质激素受体复合物选择性地与称为糖皮质激素调节元件的 DNA 序列相互作用。我们通过将受体固定在硝酸纤维素上后进行分析的程序，研究了人糖皮质激素受体与小鼠乳腺肿瘤病毒 (MMTV) DNA 之间的相互作用。将粗胞浆或核提取物中的蛋白质在 NaDodSO4/PAGE 凝胶上进行电泳，浸泡在尿素缓冲液中以去除 NaDodSO4，转移到硝酸纤维素上，并在 5% 脱脂奶粉缓冲液中用切口翻译的 MMTV [32P]DNA 进行探测，从而最大限度地减少非选择性 DNA-蛋白质相互作用。我们提供的证据表明 MMTV [32P]DNA 选择性地与糖皮质激素受体相互作用。这些数据包括[3H]甲磺酸地塞米松标记带和结合的MMTV[32P]DNA在凝胶电泳系统上的共迁移；在 0°C 下用类固醇孵育的细胞的细胞质中以及在 37°C 下孵育的细胞的细胞核和细胞质中 DNA 结合活性的定位； MMTV DNA 与高度纯化的受体结合；受体下调的细胞提取物中不存在 MMTV DNA 结合活性。此外，在这些条件下分析的糖皮质激素受体表现出与含有糖皮质激素调节元件的DNA片段的选择性结合。
To exert their effects, glucocorticoid receptor complexes interact selectively with DNA sequences known as glucocorticoid regulatory elements. We have studied the interaction between human glucocorticoid receptors and mouse mammary tumor virus (MMTV) DNA by means of a procedure that permits analysis after immobilization of the receptor on nitrocellulose. Proteins from crude cytosolic or nuclear extracts were electrophoresed on NaDodSO4/PAGE gels, soaked in a urea buffer to remove NaDodSO4, transferred to nitrocellulose, and probed with nick-translated MMTV [32P]DNA in a 5% nonfat dry milk buffer, which minimizes nonselective DNA-protein interactions. We present evidence that MMTV [32P]DNA interacts selectively with the glucocorticoid receptor. These data include comigration of [3H]dexamethasone mesylate-labeled band and bound MMTV [32P]DNA on gel electrophoresis systems; localization of DNA-binding activity in the cytosol of cells incubated with steroid at 0 degrees C and in the nucleus and cytosol of cells incubated at 37 degrees C; binding of the MMTV DNA to highly purified receptor; and absence of MMTV DNA binding activity in extracts from cells whose receptor has been down-regulated. Furthermore, glucocorticoid receptors analyzed under these conditions exhibit selective binding to DNA fragments that contain glucocorticoid regulatory elements.