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An alternative explanation for the trends observed in Figs. 1, 2, 3 could involve chelation of the added ligands not with U VI), but with U IV).
A thirty minute heat pulse at 37°C also proved adequate for the production of robust GFP fluorescence in the presence of endogenous or exogenously added ligands (see below).
VP16-SXR is transcriptionally active in the absence of added ligands [ 63].
In early metazoans, these systems added ligands and receptors; examples include the conserved Wnt and Notch Delta systems (Srivastava et al. 2010; Ryan et al. 2013; Riesgo et al. 2014).
This finding suggests that under fresh serum conditions the level of constitutively activated AhR function in P20E, even in the absence of exogenously added ligands, is already very high (i.e. higher than that is found in P20C exposed to 10 nM of TCDD).
Initial U removal rates correlated with initial U VI) concentration, as shown in Fig. 4, which shows results for inoculated control experiments containing no added ligands beyond lactate.
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The observed shifts in potential depended on both the concentration and the structure of the added ligand.
Stern Volmer analysis of the tryptophan fluorescence quenching data in presence of the added ligand reveals fluorescence quenching constant (κq), Stern Volmer constant (KSV) and also the ligand protein association constant (Ka).
Enzymes, such as DNase I, are often easy to use but typically overestimate the size of the footprint (on account of their size) and generate uneven cleavage patterns in the absence of added ligand.
Insertion of a tetracysteine-motif-containing peptide at a conserved position in Lyp's catalytic domain generated a mutant enzyme (Lyp-CCPGCC) that retains activity comparable to that of wild-type Lyp in the absence of added ligand.
In all cases the predominant form of dissolved U in the experimental media was an aqueous complex of U VI) with the appropriate added ligand.
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