Kinase Assay

MIF and ASK1 interactions were tested using a cell-free ASK1 kinase enzyme system (Promega, Madison, WI), whereby ASK1 in the presence of a substrate protein, myelin basic protein (MBP), and ATP will phosphorylate MBP, converting ATP to ADP. After quenching any remaining ATP after a reaction period, the remaining ADP is converted back to ATP and coupled to a luciferase/luciferin reaction-producing light (ADP-Glo assay; Promega). The manufacturer’s protocol was followed for setting up controls and determining the half-maximal inhibitory concentration values for inhibitors. With this assay system rMIF was titrated across several dilutions in triplicate with ASK1, and the formation of ADP was subsequently measured.