The cDNAs corresponding to various receptor isoforms (Figure 7A) were cloned in the expression vector + (Invitrogen, Carlsbad, CA, USA). The vectors were used in a coupled in vitro transcription/translation assay using the TNT kit (Promega, Madison, WI, USA). Products translated in the presence of 35 S-methionine following the manufacturer's recommendations were analyzed by SDS-PAGE on a 8% gel, and the gel was fixed, dried and exposed to X-ray films. Products translated in the absence of labeled precursor were used for band-shift experiments. Band-shift assays were performed with 2 µl of in vitro translated proteins mixed with ng of 32 P-end-labeled B1a oligonucleotide containing the NBRE site (5'gagtttt aaaaggtca tgctcaatttgg3', NBRE site underlined) in 10 mM Tris pH /40 mM KCl/ % NP-40/1 mM DTT/10 ng/µl poly dI:dC/6% glycerol. Competition experiments were performed using the B1a oligonucleotide or a mutated mB1a version (5'gagtttt aagaggtca tgctcaatttgg3'). Binding reactions were carried out for 30 min on ice and loaded onto 4% non-denaturing polyacrylamide gels. Migration conditions were 150V for 2h in TBE buffer and the gels were fixed, dried and exposed to X-ray films.
Contributor(s) Written 2004-11 Louis Dallaire
Centre de Recherche, Hôpital Ste-Justine, Montréal, Canada
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Bile acids, in excess, have been associated with pathophysiology for quite some time. Given that activation of hepatic FXR synthesis has been shown to suppress bile acid synthesis, thereby reducing the overall bile acid pool, it has been suggested that FXR agonists would be useful in the treatment of disorders related to bile acid metabolism and hepatic health. Given the critical role of the liver in glucose and lipid homeostasis, it is understandable why pharmacologic regulation of FXR is expected to have benefits in the treatment of obesity and diabetes.