Figure 2.

In vitro phosphorylation of Swi6 by Hrr25. (A) Kinase assays were done on eluates from Swi6 affinity columns or from control columns (no coupled protein). The presence of Swi6 on the column resin is indicated by a “+” above the lane, whereas “−” denotes the control column with no coupled protein. The columns were loaded with extracts from either a wild-type (lanes 1–7) or hrr25Δ strain (lanes 8 and 9) as indicated above the figure (“extract applied on column”). Exogenous substrate (100 ng) was added to the kinase assays as indicated above the lanes. mbp, myelin basic protein; H1, histone H1. (B) Kinase assays were done with 12CA5 (anti-HA) immunoprecipitates from yeast cells expressing an HA–Hrr25 fusion protein (lanes 1 and 3) or cells transformed with an empty vector (lane 2). In lanes 2 and 3, 100 ng of casein and Swi6 were added to the kinase reaction as indicated by a “+.” The position of migration of phosphorylated Swi6, Hrr25, and casein are indicated on the right. Molecular weight markers are shown on the left.