Fig. 4.

Competition study with the dual-readout F² assay. Competition of nonlabeled Noxa peptide for the binding of TMR-Noxa peptide to Mcl-1 protein. Increasing concentrations of nonlabeled Noxa peptide were added to the reaction buffer containing 125 nM of TMR-Noxa peptide and 62.5 nM of Mcl-1 protein in a final volume of 5 (L. Free (125 nM TMR-Noxa peptide alone) and bound (125 nM TMR-Noxa peptide with 62.5 nM Mcl-1 protein) tracer controls were included on each plate. The TR-FRET and FP signal were measured after a 10-h incubation at room temperature. The competition effects in TR-FRET and FP measurements were expressed as percentage of control and was calculated as described in the Materials and Methods section. The data were plotted against log10 nonlabeled Noxa peptide concentrations, and the EC₅₀ were calculated using a nonlinear regression in Prism 5.0.