Figure 1. IRAK4 kinase activity was measured in a radioisotopic filter binding assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 3 mM MgCl2, 3 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: Histone H2B, 5 µg/50 µl, Recombinant IRAK4: 20 ng/50 µl.
Figure 3. Dose dependence curve of IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, increasing amounts of IRAK4 and 1.5 µM substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of IRAK4 substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, 50 ng IRAK4, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, 50 ng of IRAK4 and increasing concentrations of substrate peptide were used per reaction at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of IRAK4 kinase activity: DELFIA® data generated using Phospho-Ezrin (Thr567)/Radixin (Thr564)/Moesin (Thr558) Antibody #3141 to detect phosphorylation of IRAK4 substrate peptide (#1344) by IRAK4 kinase. In a 50 µl reaction, 50 ng IRAK4 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)