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. 2013 Jul;11(6):367–381. doi: 10.1089/adt.2013.507

Fig. 10.

Fig. 10.

Validation of the 14-3-3/Bad TR-FRET assay in a uHTS format. The uHTS assay was validated by screening a library of 51,200 compounds. Average FRET signal (A), S/B (B), and Z′ factor (C) from the uHTS were evaluated across the forty (40) 1,536-well plates. Background FRET signals from wells containing the Dy647-pS136-Bad peptide and anti–His-Eu in the absence His-14-3-3ζ is shown in (A). (D) Summary of uHTS results. Percent inhibition of the 14-3-3ζ/pBad peptide interaction was plotted against compound fluorescence, expressed as fluorescence intensity (FI; Eu 615 nM; fold over control (FOC) for 51,200 compounds. Positive hits from the uHTS were defined as those with % inhibition >70 and 0.5<FOC<1.5. (E) Dose-response curves of three representative positive hits. (F). Comparison of the screening results for the overlapping set of 843 compounds between the current TR-FRET (14-3-3ζ/pBad peptide) assay with those from the FP (14-3-3γ/pRaf peptide) assay.