XB-IMG-181080
Xenbase Image ID: 181080
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Fig. 2. The dnN-CoRs derepress a T3-repsonse promoter by binding to TR and blocking endogenous N-CoR binding in the reconstituted frog oocyte system. (a) The dimeric dnN-CoR relieves reporter gene repression by unliganded TR more effectively than the monomeric dnN-CoR. The mRNAs for FLAG-TRα/RXR and myc-tagged dnN-CoRs (myc-ID dimer or monomer) were injected into the cytoplasm of the frog oocytes. The firefly luciferase reporter vector together with the control Renilla luciferase plasmid was then injected into the nucleus. After overnight incubation with or without T3, the oocytes were lysed and assayed for luciferase activities. The ratio of firefly luciferase activity to Renilla luciferase activity was determined as a measure of the reporter gene transcription level. The result from each group was expressed as a percentage of the basal transcription level that was obtained from the oocytes without TRα/RXR mRNA injection. The experiment was repeated 8 times and the combined results are presented here. The same oocyte samples used in luciferase assay were subjected to Western blotting with anti-myc and anti-FLAG antibodies. Representative results were shown in the lower panels, confirming the expression of dnN-CoRs and FLAG-TRα. Note that both dnN-CoRs relieved the unliganded TR-induced repression, but the derepression was more potent with the myc-ID dimer (left graph, ∗p < 0.05). Neither dnN-CoR affected the liganded-TR induced reporter gene activation (right graph). (b) myc-ID dimer interacts with unliganded TRα in frog oocytes. The mRNAs for FLAG-TRα/RXR and myc-ID dimer were injected into the cytoplasm of oocytes as indicated. After overnight incubation with or without 100 nM T3, the oocytes were lysed and subjected to immunoprecipitation (IP) with anti-FLAG antibody against TR. Pre-IP lysates and IP samples were immunoblotted with anti-FLAG and anti-myc antibodies. Note that myc-ID dimer was co-immunoprecipitated with FLAG-TRα in the sample without T3 treatment (lane 3) but not in the T3-treated sample (lane 4). (c) myc-ID dimer competes with the endogenous N-CoR for binding to unliganded TR. The mRNAs for FLAG-TRα/RXR and myc-ID dimer were injected into the cytoplasm of oocytes as indicated. After overnight incubation with or without 100 nM T3, the oocyte lysates were subjected to IP with anti-FLAG antibody. Pre-IP lysates and IP samples were immunoblotted with anti-FLAG, anti-N-CoR, and anti-myc antibodies. Similar to myc-ID dimer, endogenous N-CoR was co-immunoprecipitated with FLAG-TRα only in the sample without T3 treatment (compare lane 2 and 3 in the second panel). The myc-ID dimer overexpression reduced the amount of endogenous N-CoR co-immunoprecipitated with FLAG-TRα (compare lane 2 and 4 in the second panel). The experiments in Fig. 2 were repeated at least 3 times with similar results. Image published in: Sato Y et al. (2007) Copyright © 2007. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |