Figure 1. PDGFRalpha D842V kinase activity was measured in a radiometric assay using the following reaction conditions: 60 mM HEPES-NaOH, pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 µM Na-orthovanadate, 1.2 mM DTT, 100 µM ATP, 100 µM FLT3 (Tyr589) Biotinylated Peptide (#1305) and variable amount of Recombinant PDGFRalpha D842V. Reaction mixture incubated at room temperature for 10 minutes.
Figure 3. Dose dependence curve of PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, increasing amounts of PDGFRalpha D842V 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. Sensitivities of wild type and mutant PDGFRalpha D842V kinases to Staurosporine (A) and Gleevec (B) were compared: DELFIA® data generated using Phospho-Tyrosine mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha substrate peptide (#1305) by the PDGFRalpha kinases. In a 50 µl reaction, 100 ng of kinase, 1.5 µM substrate peptide, 20 µM ATP and increasing concentration of indicated inhibitors were used per reaction well at room temperature for 30 minutes. (DELFIA® is a registered trademark of PerkinElmer, Inc.)
Figure 4. Peptide concentration dependence of PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, 100 ng of PDGFRalpha D842V 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 PDGFRalpha D842V kinase activity: DELFIA® data generated using Phospho-Tyrosine Mouse mAb (P-Tyr-100) #9411 to detect phosphorylation of PDGFRalpha D842V substrate peptide (#1305) by PDGFRalpha D842V kinase. In a 50 µl reaction, 100 ng PDGFRalpha D842V and 1.5 µM substrate peptide were used per reaction. (DELFIA® is a registered trademark of PerkinElmer, Inc.)