4.8. IRAK1 and IRAK4 Kinase Assay

To measure the ability of Dt-EE to inhibit the activity of the IRAK1 and IRAK4 enzymes, we used a kinase profiler service from Millipore (St. St. Louis, MO, USA). IRAK1 and IRAK4 (human) (1–5 mU) were incubated with a reaction buffer for a final reaction volume of 25 µL, and the reaction was started by adding MgATP. The reacted samples were incubated at room temperature for 40 min, and then the reaction was completed by adding 5 mL of 3% phosphoric acid solution. 10 µL of the reaction products were then spotted onto a P30 filtermat and washed three times for 5 min in 75 mM phosphoric acid and once in methanol before drying and scintillation counting.