Download Constitutive Activity in Receptors and Other Proteins, Part by P. Michael Conn (Eds.) PDF

By P. Michael Conn (Eds.)

This quantity of equipment in Enzymology covers the present method for the detection and evaluate of constitutively lively proteins. The chapters written by way of specialist authors who're leaders within the field, provide tricks and tips now not to be had in fundamental examine publications.It is generally referenced, with beneficial figures and tables in the course of the volume.

A. professional authors who're leaders within the field

B. broadly referenced and important figures and tables

C. offers tricks and tips to facilitate copy of methods

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Additional info for Constitutive Activity in Receptors and Other Proteins, Part B

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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.

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