Figure 4.
In vitro activation of Cdc28p-HA. (A) Beads containing Cdc28p-HA immunoprecipitated from insect cells were phosphorylated in vitro with GST-Cak1p (PO4-Cdc28p-HA) or left untreated (Cdc28p-HA). The indicated amounts of beads were immunoblotted with the α-P-Thr-169 (top) and α-HA (bottom) antibodies. The smear in the 20-μl lane of the α-P-Thr-169 blot is due to IgG present in the immunoprecipitates. (B) Cdc28p-HA beads were incubated with MBP-Clb2p (Clb), MBP-Cln2p (Cln), GST-Cak1p, and Cks1p in the indicated combinations. Beads were washed five times with 200 μl of buffer K and then assayed for histone H1 kinase activity. Activity was quantitated with the use of a phosphorimager, and the relative activity (rel. act.) is listed beneath each lane, with the lowest activity normalized to 1.