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Fig. 5. GST-Flp1p dephosphorylates in vitro CDK-phosphorylated GST-Cdc25p. Purified GST-Flp1p and GST-Flp1CSp proteins from cdc25{Delta} cdc2-3w strains (as in Fig. 4A) were assayed in vitro, in the linear range of the assay, for their ability to dephosphorylate purified GST-Cdc25p from bacteria and previously phosphorylated in vitro by immunoprecipitated Cdc2/Cdc13 complexes. The relative amount of radioactive GST-Cdc25p was quantified in three different and independent experiments (in which every component was de novo purified or immunoprecipitated) and plotted. 110 µM CDK-phosphorylated GST-Cdc25p were incubated with 0 µM (sample 1 and 6), 55 µM (sample 2), 110 µM (sample 3) or 220 µM (sample 4) of GST-Flp1p or 330 µM GST-Flp1CSp (sample 5). Samples were run on PAGE gels and stained with Coomassie Blue. Relative phosphorylation levels in GST-Cdc25p were quantitated using a phosphorimager and plotted as a percentage of the phosphorylation levels measured/observed in reactions without GST-Flp1p. Note that the extent of GST-Cdc25p dephosphorylation does increase slightly with addition of more GST-Flp1p. Bars indicate standard error.





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