Figure 1. ROCK2 kinase activity was measured in a radiometric assay using the following reaction conditions: 5 mM MOPS, pH 7.2, 2.5 mM β-glycerophosphate, 1 mM EGTA, 0.4 mM EDTA, 5 mM MgCl2, 0.05 mM DTT, 50 μM ATP, Substrate: S6K substrate peptide (KRRRLASLR) 400 ng/μL, and variable amounts of recombinant ROCK2.
Figure 3. Dose dependence curve of ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, increasing amounts of ROCK2 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of ROCK2 substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng ROCK2, 1.5 µM substrate peptide, 50 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng of ROCK2 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 ROCK2 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibodty #5070 to detect phosphorylation of ROCK2 substrate peptide (#1300) by ROCK2 kinase. In a 50 µl reaction, 50 ng ROCK2 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)