Figure 1. PKCepsilon kinase activity was measured in a radiometric assay using the following reaction conditions: 4 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 4 mM MgCl2, 0.05 mM DTT, 40 ng/μl BSA, 50 μg/ml phosphatidylserine, 5 μg/ml diacylglycerol, 0.1 mM sodium orthovanadate, 0.1 mM dithioreitol, 0.1 mM CaCl2, 50 μM ATP, Substrate: PKCepsilon substrate peptide 300 ng/μL and recombinant PKCepsilon: variable.
Figure 3. Dose dependence curve of PKCepsilon kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCepsilon kinase. In a 50 µl reaction, increasing amounts of PKCepsilon 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 PKCepsilon kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCepsilon substrate peptide (#1331) by PKCepsilon kinase. In a 50 µl reaction, 25 ng PKCepsilon, 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 PKCepsilon kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of substrate peptide (#1331) by PKCepsilon kinase. In a 50 µl reaction, 25 ng of PKCepsilon 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 PKCepsilon kinase activity: DELFIA® data generated using Phospho-PKA Substrate (RRXS/T) (100G7) Rabbit mAb #9624 to detect phosphorylation of PKCepsilon substrate peptide (#1331) by PKCepsilon kinase. In a 50 µl reaction, 25 ng PKCepsilon and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)