FIG. 6.

BUR2 is required for Bur1 kinase activity. Extracts were prepared from either BUR2⁺ (GY458) or bur2Δ (GY832) strains transformed with the 2μm plasmids pSM21 (FLAG-BUR1), pSM14 (FLAG-bur1-3), and pSY14 (BUR2) in the combinations indicated at the top of panel A. Proteins were immunoprecipitated with FLAG-conjugated agarose beads and washed extensively, and [γ-³²P]ATP was added to detect kinase activity. (A) Kinase assay. An autoradiogram of the SDS-polyacrylamide gel is shown, with size markers indicated in kilodaltons on the left. The two major phosphorylated proteins of >200 and 80 kDa that are specific for the FLAG-BUR1 extract are indicated by the arrows on the right. (B) Western blot. Proteins immunoprecipitated in the assay in panel A were probed with an anti-FLAG monoclonal antibody. Arrows indicate the FLAG-Bur1 (lanes 2, 4, and 5) and FLAG-bur1-3 (lane 3) bands.