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. 2000 May;11(5):1597–1609. doi: 10.1091/mbc.11.5.1597

Figure 5.

Figure 5

Dependence of Cdc28p activity on the order of addition of cyclin and Cak1p. Beads containing Cdc28p-HA immunoprecipitated from insect cells were incubated with MBP-Clb2p (Clb), MBP-Cln2p (Cln), and GST-Cak1p (Cak) as indicated for 30 min (first incubation [1st inc.]). Beads were washed five times with 200 μl of buffer K and then incubated for 30 min with MBP-Clb2p (Clb), MBP-Cln2p (Cln), and GST-Cak1p (Cak) as indicated (second incubation [2nd inc.]). Beads were again washed five times with 200 μl of buffer K and finally assayed for histone H1 kinase activity. All samples contained 100 nM Cks1p and 500 μM ATP during the first and second incubations. 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.