Figure 1. Akt3 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: GSK3 (14-27), 5 µg/50 µl, recombinant Akt3: 200 ng/50 µl.
Figure 3. Dose dependence curve of Akt3 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of substrate peptide (#1133) by Akt3 kinase. In a 50 µl reaction, increasing amounts of Akt3 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 Akt3 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of Akt3 substrate peptide (#1133) by Akt3 kinase. In a 50 µl reaction, 50 ng Akt3, 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 Akt3 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of substrate peptide (#1133) by Akt3 kinase. In a 50 µl reaction, 50 ng of Akt3 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 Akt3 kinase activity: DELFIA® data generated using Phospho-eNOS (Ser1177) Antibody #9571 to detect phosphorylation of Akt3 substrate peptide (#1133) by Akt3 kinase. In a 50 µl reaction, 50 ng Akt3 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)