Figure 3. Ipl1 activation by Sli15 is independent of Sli15 phosphorylation.

GST-Ipl1 (Ipl1), GST-Sli15 (Sli15), recombinant PP1γ and [γ-³²P]ATP were pre-incubated for 20 min, a time that allowed maximal phosphorylation of Sli15 in the absence of PP1γ, in either the presence or absence of the PP1 inhibitor microcystin. A sample (P) was taken, then microcystin was added to the reaction that lacked the inhibitor and myelin basic protein (MBP) added to both reactions, taking further samples over a 15-minute time course. All samples were separated by SDS-PAGE and both MBP and Sli15 phosphorylation monitored by autoradiography. (A) Schematic showing the experimental design. (B) Autoradiograph. (C) Quantitation of Sli15 and MBP bands shown in (B) by liquid scintillation counting after excising the labelled bands.