In vitro kinase assay

Catalytically active rhTAK1-TAB1 or other protein kinases were reacted with major basic protein (MBP, 0.33 mg ml⁻¹) as an exogenous substrate and [γ-³²P]ATP (5 μCi) as the probe for 30 min at 30 °C. The reaction mixtures were spotted onto a P81 phosphocellulose filter, and washed extensively with 0.8% H₃PO₄ followed by 98% acetone. Radioactivity on the filter was measured as count per min (cpm). rhTAK1-TAB1-catalyzed kinetic parameters, Michaelis–Menten K_m constant and maximal velocity (V_max), were determined by the Lineweaver–Burk plots.