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Analysis of enzymatic activity was performed according to the Zlyte assay protocol (Invitrogen):
1. Different concentrations of PAK1 were incubated in a buffer containing 50 mM HEPES pH 7.5, 10 mM MgCl2, 1 mM EGTA, 200 uM ATP, 0.01% Brij-35, and 2 uM substrate (SER/THR 14, Invitrogen) at RT for 1 hour.
2. Developer solution was added to the reaction and the reaction was stopped after 1 hour of incubation at RT.
3. Fluorescence was then detected using λexc=460±40 nm and λem=528±20 nm filters.