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Additional info for Constitutive Activity in Receptors and Other Proteins, Part B
2. On the day of transfection Quantities and volumes of reagents are intended for transfection of each T-125 flask. 1. 2 mL of serum-free Ham’s F-12 medium and mixed thoroughly by vortexing. 2. Incubation at room temperature was performed for 20 min. 3. 1 plasmid (Invitrogen) were added to the diluted TransIT-CHO reagent and mixed by gentle pipetting. 1 plasmids alone, as opposed to “þNR” cells, which were cotransfected with the Gal4-LBD encoding plasmids. 4. Incubation at room temperature was performed for 20 min.
For this reason, reporter genes harboring Gal4 binding sites have very low background expression when transfected in mammalian cells (Kakidani and Ptashne, 1988). Primers GATCGGATCCCCGGAGCCTTATGCCAGCCC (forward) and GATCTCTAGATCAAGTCTGCTTGGCTTGCAGCAT TTCGATGAG (reverse), harboring a BamHI and a XbaI site, respectively (underlined sequence), were used to amplify the SF-1 LBD (aa. 198–462) cDNA using an expressed sequence tag (EST) clone (Invitrogen) as a template. The RORA LBD (aa. 266–523) cDNA was generated by PCR using primers GCCGCCCCCGGGCCGAACTAGAACACCTTGCCC (forward) and TATATAAAGCTTTCCTTACCCATCGATTTGCAT GG (reverse), harboring a XmaI and a HindIII site, respectively (underlined sequence), and a mouse liver cDNA library (Clontech) as a template.
49, 6652–6655. Wilson, T. , Fahrner, T. , and Milbrandt, J. (1993). The orphan receptors NGFI-B and Steroidogenic Factor 1 establish monomer binding as a third paradigm of nuclear receptor-DNA interaction. Mol. Cell. Biol. 13, 5794–5804. Winnay, J. , and Hammer, G. D. (2006). Adrenocorticotropic hormone-mediated signaling cascades coordinate a cyclic pattern of Steroidogenic Factor 1-dependent transcriptional activation. Mol. Endocrinol. 20, 147–166. Zhang, J. , Chung, T. , and Oldenburg, K. R.