Supplementary Figure 1
From: Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis
(A) Michaelis-Menten kinetic parameters of purified PP2A-B55α holoenzyme were determined against 16-mer phospho-peptides originating from Cdh1 144-158 (pS151: WDVSPYSL(pS)PVSNKSQ; pT151 WDVSPYSL(pT)PVSNKSQ) using malachite green phosphatase assay. Mean and standard deviation shown in plot (n = 3 independent experiments). (B) Endogenous Cdc20 was depleted by RNAi in HeLa cells using the outlined protocol and complemented with the indicated YFP tagged forms of Cdc20 and mitotic duration was determined using live cell microscopy (n values are shown in the graph and represent single cells analyzed per condition, Median and interquartile range indicated by red lines from 2 independent experiments). Representative still images are shown below. Scale bar; 10 μm. (C) Mitotic APC/C devoid of Cdc20 was incubated with the indicated purified Strep tagged Cdc20 proteins. After washing the amount of Cdc20 remaining/bound was determined by western blot.
