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Fig. 8 Characterization of the retinoic-acid-responsive Wt1 enhancer. (A) Electrophoretic mobility shift assay (EMSA) using in vitro transcription/translation (TNT) products of RARβ and RXRα on the zebrafish (left) and the human (right) enhancer. TNT extract programmed with the empty Rc/CMV vector was used as a negative control. Mutant fragments for competition experiments harbored a deletion of conserved region 1 (cons 1) in the background of the zebrafish or human enhancer. For supershifts, an antibody against RARβ or control antiserum was used. (B) Supershift assays using the human WT1 enhancer fragment as the probe. Nuclear extracts were prepared from P19 cells treated with RA (+) or ethanol (-) for 24 hours. Analysis was performed using antibodies specific for RARβ and RARα or respective control antibodies. The different mobility of the RARβ and RARα supershift complexes is most likely to be explained by the antibodies used (monoclonal versus polyclonal antiserum). Note that pre-incubation of the extracts with RARβ antiserum resulted in either immunodepletion (A) or supershift (B) of the DNA-protein complexes, depending on the type of extract. (C) Schematic luciferase reporter constructs. The sequence of the inserted human WT1 enhancer region (287 bp) and of the deleted conserved elements (cons 1-3) is shown in Fig. 5B. (D) P19 cells were transfected with the constructs shown in A and grown for 4 days in the presence of 1 μM all-trans retinoic acid (atRA) or vehicle (0.1% ethanol). Subsequently, cells were lysed and luciferase activity was measured. Luciferase expression in vehicle-treated cells transfected with pGL3 vector was set to one. The figure shows one representative experiment. Data represent the means and standard deviations of triplicates. Asterisks indicate statistically significant differences, *P<0.05, Student′s t test. mut, mutant; wt, wild type.