Xenbase Image ID: 151122

Figure 1. Apparent dissociation constant (Kd) analysis in vivo using three different concentrations of specific DNA sites in Xenopus oocytes. (A) Experimental design. (B) The pBS(HRE)x7 harbors seven repeats of the DNA segment, the partially palindromic HRE with its two binding elements (BE) highlighted in gray and a FoxA1 binding site is indicated by a rectangle. The protected guanines within the HRE are highlighted by empty circles and the direction of primer extension is shown by a black arrow. (C) Upper diagram, analysis of amount of [3H]-Dex per oocyte nucleus in double samples after oocyte injection with indicated amount of GR mRNA (ng). Four samples were incubated with [3H]-Dex supplemented with excess of unlabeled Dex. Stippled line indicates background that was subtracted. Lower diagram: nuclear GR concentration as a function of GR mRNA injection. (D) Autoradiogram showing the pattern of dimethylsulphate (DMS) methylation at increasing GR concentrations using 0.05 μM of HRE as shown in 1E, double samples for each GR concentration. Gray arrows show borders of the seven repeats, The HRE is indicated by two gray boxes with the protected bands as empty circles for the first repeat (to the right). Scans and columns illustrate quantification of methylation of corresponding guanines with error bars showing the average deviation of double samples. (E) GR-HRE binding analyzed by DMS methylation protection for three different concentrations of HRE as determined by primer extension (See also Supplementary Figure S1A). The concentration of nuclear GR required for 50% GR-HRE binding or DMS methylation protection, is highlighted by arrows pointing at the x-axis. The 50% binding of the 0.5 μM GRE curve was extrapolated from 47.2% binding at the highest GR concentration (dotted line). (F) Graphic estimation of Kd: when total nuclear GR concentration at 50% HRE binding is plotted as a function of the concentration of GR:HRE complex at 50% saturation then the free GR concentration present in absence of any GR:HRE complex is equal to the apparent Kd. Image published in: Belikov S et al. (2016) © The Author(s) 2015. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial license Larger Image Printer Friendly View

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