Tracer addition: 20X K10 tracer (Promega #N264B) in tracer dilution buffer (Promega #N219B) was prepared from 100X of K10 in DMSO to yield final assay concentrations of 25 nM, 100 nM, 250 nM, and 1 µM for the respective kinases. 20X K10 (5 µL) was added to each well, excluding background wells and the bottom control wells (zero BRET). Assay plates were mixed on an orbital shaker for 15 seconds at 300 rpm.