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• W1566966041 abstract "This chapter focuses on the methods for detecting domain interactions in nuclear receptors. Amino- and carboxyl-terminal (N–C) domain interactions in the human androgen receptor (AR) are demonstrated in the studies based on the dissociation rate of bound ligand. An N–C interaction in the AR is also supported by the finding that NH2- and carboxyl-terminal fragments of the receptor, each containing the DNA-binding domain, exhibited androgen-dependent DNA binding and dimerization. Detection methods for the AR N–C interaction have changed over the years to optimize transfection efficiency and signal intensity and to facilitate tests for specific interacting sequences. Instead of the Chinese hamster ovary (CHO) cells used originally in the assay—humanepithelioid cervical carcinoma (HeLa) or human hepatocellular carcinoma (HepG2) cells—are employed. The Effectene TM reagent from Qiagen is used for transient transfection rather than DEAE dextran. The G5E1bLuc reporter vector is replaced by 5XGAL4Luc3, which yields a stronger signal. An additional method to confirm protein–protein interaction sites among nuclear receptor sequences involves determining the dissociation rate of bound radiolabeled ligand from chimeric receptor proteins. Another approach to demonstrate protein–protein interactions is GST affinity matrix or pull-down assays. GST affinity matrix assays demonstrate protein interactions, but it must be kept in mind that nonspecific binding can occur when high concentrations of protein are used. In studies with nuclear receptors, this potential complication is minimized by demonstrating hormone dependence for the interaction." @default.
• W1566966041 created "2016-06-24" @default.
• W1566966041 creator A5012199796 @default.
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• W1566966041 date "2003-01-01" @default.
• W1566966041 modified "2023-10-16" @default.
• W1566966041 title "Methods for Detecting Domain Interactions in Nuclear Receptors" @default.
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• W1566966041 doi "https://doi.org/10.1016/s0076-6879(03)64008-5" @default.
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• W1566966041 hasPublicationYear "2003" @default.
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