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. Author manuscript; available in PMC: 2010 May 14.
Published in final edited form as: Mol Cell. 2009 May 14;34(4):497–509. doi: 10.1016/j.molcel.2009.04.011

Figure 5. 14-3-3 proteins bind PACS-2 phospho-Ser437.

Figure 5

(A) PACS-2 was immunoprecipitated from rat brain cytosol and co-precipitating 14-3-3 was detected by western blot. (B) The indicated GST fusion proteins were incubated with ATP and constitutively active HA-Akt (WT) or kinase-dead HA-Akt (KD), then incubated with 14-3-3σ-his6. Protein complexes were captured with glutathione-sepharose and bound 14-3-3σ-his6 was detected by western blot. (C) (TMR)–labeled 16mer peptides containing pSer259-Raf, PACS-2 Ser244-PACS-2, pSer244-PACS-2, Ser437-PACS-2 or pSer437-PACS-2 (1 nM) were incubated in triplicate with increasing concentrations of GST-14-3-3γ. Fluorescence polarization signals were recorded and used to calculate Kds. (D) 14-3-3 isoforms were incubated with (TMR)-labeled pSer437-PACS-2 or pSer259-Raf peptides and analyzed as described in panel C. Calculated Kd values are shown.