Fig. (5) AlphaScreen assay for measuring the interaction of 14-3-3γ with pS259-Raf-1. Experiments were performed as described in the legend to Fig. (4) using 100 nM of GST-14-3-3γ and 100 nM of biotin-pS259-Raf-1 peptide. For (A), (D), and (E), data are expressed as means ± SD from triplicate determinations. (A) Increasing concentrations of GST-14-3-3γ protein were incubated with biotin-pS-259-Raf peptide at RT for 30 min. Donor and acceptor beads were added with an interval of 30 min. Results were recorded after 3 hr of incubation at RT. (B) The S/B ratios of the assay. (C) The Z’ factor of the assay. (D) Competition with the R18 peptide or its mutant peptide, R18Lys, in the 14-3-3γ/pS259-Raf-1 AlphaScreen assay. (E) Competition assay using the pS967-ASK1 or its non-phosphorylated ASK1 control peptide.