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. 2013 Dec 31;8(12):e84569. doi: 10.1371/journal.pone.0084569

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© 2013 Mulero et al

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) Effect of polydIdC addition on RXR/PPARG2 and RXR/THR BRET assay. A BRET shift assay in PLB was performed in presence or absence of polydIdC (50 ng/µl) and effect on basal BRET and specific DNA RE induced BRET shift was measured for RXR-Luc and PPARG2-EYFP (**P<0.01; ***P<0.001), or RXR-Luc and THR-EYFP (#P<0.05; ###P<0.001), Statistical differences relative to control (TE) were analyzed by one-way ANOVA followed by Dunnett’s multiple comparison post hoc test. (B, C and D) Effect of MgCl₂ addition on BRET shift RXR/PPARG2, RXR/THR and RXR/RXR BRET assay. BRET shift experiments were carried out in Gel Shift Buffer (GSB) supplemented with 0, 2 or 5 mM of MgCl₂. Calculation of the DR1/DR4 BRET shift ratio for RXR/PPARG2 (B), the DR4/DR1 BRET ratio for RXR/THR (C) and the DR1/DR4 BRET ratio for RXR/RXR (D), illustrated the higher specificity of BRET shift observed by increasing MgCl₂ concentration. Histograms represent mean BRET value ± SD of at least 3 experiments in triplicate. Values represent BRET measures integrated over a 20 min reading.