Competition assays with unmarked RNA oligonucleotide and with rTTP protein.

<p><b>A</b>) The percentage of inhibition increased as function of untagged TNFα RNA probe (U-TNF) concentration. U-TNF was added to the reaction together with Bi-TNF probe and signals were detected at equilibrium. <i>Ki</i> values were determined from nonlinear regression fits of the data according to 1-site fit <i>Ki</i> model in GraphPad Prism®, version 5.0, by keeping constant the concentration (50 nM) and the <i>Kd</i> (2.5 nM) for the labeled Bi-TNF probe. Mean and standard deviation values derive from two independent experiments. <b>B</b>) Coomassie staining of purified and recovered Zeba^TM Spin desalted rTTP protein (25 ng) loaded on 15% SDS-PAGE. <b>C</b>) REMSA showing rTTP (0.1 µM) complexed with Cy-TNF RNA probe (0.5 µM reacted and loaded on native gel). <b>D</b>) Competitive AlphaScreen assay as a function of rTTP concentration. Equal amounts of BSA were independently reacted as negative control. <i>Ki</i> values were determined from nonlinear regression fits of the data according to 1-site fit <i>Ki</i> model in GraphPad Prism®, version 5.0, by keeping constant the concentration (50 nM) and the <i>Kd</i> (2.5 nM) for the labeled Bi-TNF probe. Mean and standard deviation values derive from two independent experiments with two rTTP protein preparations. <b>E</b>) Western blot showing rHuR purified from control (Mock; DMSO) and CsA [4 µM] stimulated HEK293T cells. After 3 hr of treatment the total amount of purified rHuR proteins (150 ng loaded on gel) was not affected, while the phosphorylation of the protein was clearly induced, as showed by an anti-phosphoserine antibody (P-SER). <b>F</b>) Saturation binding experiments comparing the binding capability of rHuR and phosphorylated rHuR (P-rHuR). Nonlinear regression fits of the data revealed an equilibrium dissociation constants equal to 3.1±0.55 nM for P-rHuR, not statistically relevant (<i>P value</i> = 0.59) with respect to the <i>Kd</i> of rHuR. Mean and standard deviation values derive from two independent experiments.