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. 2008 Nov 6;2:40–47. doi: 10.2174/1875397300802010040

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© Du et al.; Licensee Bentham Open.

This is an open access article licensed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted, non-Commercial use, distribution and reproduction in any medium, provided the work is properly cited.

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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.