Fig. 6.

Validation of the 14-3-3/pS136-Bad interaction assay with known 14-3-3 inhibitors. An increasing concentration of 14-3-3 peptide antagonists or a small-molecule inhibitor was added to a reaction containing the Dy647-pS136-Bad peptide (20 nM), His-14-3-3ζ (10 nM), and anti–His-Eu (1 nM). After incubation at room temperature for 2 h, the TR-FRET signals were recorded. The effect of the 14-3-3 inhibitors on the TR-FRET signal is expressed as % of control as described in the Materials and Methods section. The effect of the pS976-ASK1 peptide, or a nonphosphorylated ASK1 peptide (A), the pS259-Raf-1 peptide (B), R18 peptide (C), or FOBISIN101 (D), on the TR-FRET signals from the 14-3-3/pS136-Bad interaction is shown.