Figure 1. SGK3 kinase activity was measured in a radioisotopic filter binding 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, 1 µM ATP, 2.5 µg/50 µl PEG20,000, Substrate: GST-GSK3 (14-27), variable and recombinant SGK3, variable.
Figure 3. Dose dependence curve of SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, increasing amounts of SGK3 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 SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of SGK3 substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng SGK3, 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 SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng of SGK3 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 SGK3 kinase activity: DELFIA® data generated using Phospho-PLK (Ser137) Antibody #5070 to detect phosphorylation of SGK3 substrate peptide (#1300) by SGK3 kinase. In a 50 µl reaction, 50 ng SGK3 and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)