Characterization of the functional binding of rHuR to the AU-rich RNA substrate.

<p><b>A</b>) REMSA showing the binding capability of rHuR (0.5 µM) resulting in the presence of an higher molecular weight protein-RNA complex with respect to the free Cy-TNF RNA probe (0.5 µM). The supershift caused by the anti-HuR antibody (1 µg) indicates the presence of at least a ternary complex and the qualitative binding of rHuR. <b>B</b>) Saturation binding experiments. Equilibrium dissociation constants (<i>Kd</i>) were determined from nonlinear regression fits of the data according to a 1-site binding model in GraphPad Prism®, version 5.0. Mean and standard deviation values derive from four independent experiments with four rHuR protein preparations. <b>C</b>) Time course experiments. Association (<i>K</i>_<i>on</i>) and dissociation (<i>K</i>_<i>off</i>) rate constants were determined from nonlinear regression fits of the data according to association kinetic model of multiple ligand concentration in GraphPad Prism®, version 5.0. Mean and standard deviation values derive from two independent experiments with two rHuR protein preparations.