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. 2011 Feb 11;286(13):10911–10917. doi: 10.1074/jbc.C110.210211

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© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 2. — A reconstituted GAS41-PP2Cβ heterodimer binds to and dephosphorylates p53 at serine 366 in vitro. A, double affinity purification of the heterodimeric GAS41-PP2Cβ complex. pET28b-His-PP2Cβ1 (Km^R) and pGEX-GST-GAS41 (Amp^R) were coexpressed in E. coli, and the protein complexes were purified by sequential affinity purifications on Ni-NTA and glutathione-Sepharose 4B resins. The preparations were resolved on an SDS-PAGE gel and stained with Coomassie Brilliant Blue R-250. B, in vitro serine/threonine phosphatase assay of PP2Cβ1 monomer and GAS41-PP2Cβ1 heterodimer. Three independent colorimetric measurements of phosphatase assays were carried out, and the average values are shown. Free P, free phosphate. Error bars indicate S.D. C, direct binding of GAS41-PP2Cβ1 to p53 in vitro. Recombinant His-PP2Cβ1 monomer and FLAG-GAS41-His-PP2Cβ1 heterodimer, purified from E. coli, were used in GST pulldown assays. The amounts of recombinant proteins used in the assays are shown in the Coomassie Brilliant Blue R-250-stained gel (upper Input panel). His-PP2Cβ1 binding to GST (bottom panel, lane 2) or GST-p53 (bottom panel, lane 3) proteins was scored by immunoblot using anti-PP2Cβ antibody. D, selective dephosphorylation of serine 366 on p53 by the GAS41-PP2Cβ1 complex. Equal amounts of purified FLAG-p53 derived from UV-treated 293T cells (lanes 2–6) were subjected to phosphatase assays using calf intestinal phosphatase (CIP, lane 3), recombinant His-PP2Cβ1 monomer (lane 4), or two preparations of PP2Cβ1 heterodimers containing GST-GAS41 (lane 5) and FLAG-GAS41 (lane 6), respectively. Dephosphorylation of serine 366 on the p53 was monitored by immunoblot with a phosphospecific p53 antibody. E, dephosphorylation assay of p53 at serine 15. Equal amounts of purified FLAG-p53 derived from UV-treated 293T cells (lanes 1–5) were subjected to phosphatase assays with calf intestinal phosphatase (lane 1), recombinant His-PP2Cβ1 monomer (lane 3), or two preparations of PP2Cβ1 heterodimers containing GST-GAS41 (lane 4) and FLAG-GAS41 (lane 5), respectively. Dephosphorylation of serine 15 on the p53 was monitored by immunoblot with a phosphospecific p53 antibody.