Figure 3
From: The isothiocyanate class of bioactive nutrients covalently inhibit the MEKK1 protein kinase
AMP-PNP interferes with PEITC inhibition of MEKK1. MEKK1 catalytic domain fragment or the C1238V mutant were purified on chitin beads and then preincubated with 2 mM AMP-PNP/10 mM MgCl2 in 50 mM Tris pH7.8 for 15 minutes at room temperature. PEITC was added to the reactions, and the incubations continued for an additional 30 minutes. The bead bound proteins were then washed to remove the agents and kinase activity measured. Preincubation with AMP-PNP protected MEKK1 from inhibition by subsequent exposure to PEITC in vitro. The C1238V mutant was unaffected by any of the treatments.