Figure 1. IKKbeta kinase activity was measured in a radiometric 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: Rb CTF 1.5 µg/50 µl, recombinant IKKbeta: 50 ng/50 µl.
Figure 3. Dose dependence curve of IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, increasing amounts of IKKbeta 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 IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of IKKbeta substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng IKKbeta, 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 IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng of IKKbeta 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 IKKbeta kinase activity: DELFIA® data generated using Phospho-IkappaB-alpha (Ser32/36) (5A5) Mouse mAb #9246 to detect phosphorylation of IKKbeta substrate peptide (#1146) by IKKbeta kinase. In a 50 µl reaction, 50 ng IKKbeta and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)