Figure 2.

GST-Mob1p precipitates Mps1p protein kinase. (A) Expression and purification of GST-Mob1p from yeast (see MATERIALS AND METHODS). Lane 1, total yeast protein containing expressed GST-Mob1p stained with Coomassie brilliant blue on SDS-PAGE. Lane 2, GST-Mob1p purified from yeast extract on glutathione-Sepharose. Positions of molecular weight markers are shown. (B) Protein from yeast extract containing myc-epitope- tagged Mps1p was precipitated with anti-myc antibody (MYC IP), GST-Mob1p-bound Sepharose (GST-Mob1p), or GST-bound Sepharose (GST). CBB, Coomassie brilliant blue-stained gel of precipitants. Kinase assay, autoradiogram showing ³²P-labeled proteins in corresponding precipitants after performance of an Mps1p protein kinase assay. Immunoblot, precipitated protein were immunoblotted and probed with 9E10. The prominent ∼ 60 kDa band below myc-Mps1p on the immunoblot of MYC-IP is IgG heavy chain of the primary antibody recognized by the secondary antibody. Note that active Mps1p protein kinase is present in the anti-myc and GST-Mob1p precipitants but not in the GST precipitants. Also note that in addition to myc-Mps1p autophosphorylation, GST-Mob1p is phosphorylated in vitro. Molecular weight markers (M) and the positions of GST, GST-Mob1p, and myc-Mps1p are shown.