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Figure 3


Fig. 3. Plo1p binds directly to and phosphorylates Mbx1p in vitro. (A) In vitro kinase assay with purified 6His-Plo1p and recombinant components of PBF. Separate kinase reactions with 6His-Plo1p and either 6His-Fkh2p, GST-Sep1p or GST-Mbx1p were separated by SDS PAGE, stained with Coomassie Blue and autoradiographed. Arrow indicates phosphorylated GST-Mbx1p, near to autophosphorylated 6His-Plo1p. Table indicates combinations of tagged proteins used. (B) In vitro kinase assay with purified 6His-Plo1p and GST-Mbx1p bound to glutathione-Sephadex beads after washes with 1% NP-40 to remove 6His-Plo1p, separated by SDS PAGE, stained with Coomassie Blue and autoradiographed. Arrow indicates phosphorylated GST-Mbx1p. Table indicates combinations of tagged proteins used.