Figure 1. PKD/PKCmu kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 1.32 mM CaCl2, 1 mM EDTA, 1.25 mM EGTA, 0.25 μg/50 μl phosphatidylserine, 50 ng/50 μl 1.2 Dioleyl-glycerol, 1.2 mM DTT, ATP (variable), 2.5 µg/50 µl PEG20.000, Substrate: tetra (LRRWSLG), 0.5 µg/50 µl and recombinant PKD/PKCmu: 200 ng/50 µl.
Figure 3. Dose dependence curve of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, increasing amounts of PKD/PKCmu and 1.5 µM substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 5. Staurosporine inhibition of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKD/PKCmu substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng PKD/PKCmu, 1.5 µM substrate peptide, 20 µM ATP and increasing amounts of staurosporine were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng of PKD/PKCmu and increasing concentrations of substrate peptide were used per reaction at room temperature for 15 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 2. Time course of PKD/PKCmu kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKD/PKCmu substrate peptide (#1331) by PKD/PKCmu kinase. In a 50 µl reaction, 10 ng PKD/PKCmu and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)