2.9. In vitro kinase assay

The FAK Kinase Enzyme System (V9301, Promega) was used for in vitro kinase enzymatic assay. The effect of M64HCl or M64 freebase on FAK activity was measured using a luminescent assay. The ADP-Glo™ Assay is a luminescent ADP detection assay that provides a homogeneous, high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction. After the kinase reaction, the detection assay is performed in two steps; first, an equal volume of ADP-Glo™ Reagent is added to terminate the kinase reaction and deplete the remaining ATP. Second, the Detection Reagent is added to simultaneously convert ADP to ATP and allow the newly synthesized ATP to be measured using a luciferase/luciferin reaction. The light generated is measured using a luminometer. The luminescent signal positively correlates with kinase activity. Luminescence was measured using a Tecan Spark plate reader. (Grödig, Austria).