Staurosporine inhibition of enzyme activity. A concentration of 8.6 nM PKCα was used to phosphorylated Histone H1 or PHAS-1 for 60 minutes at ~25°C using various concentrations of Staurosporine (3A). Inhibition of PKCα and IRAK4 activities using MBP as a substrate were performed using decreasing concentrations of Staurosproine (3B). Following incubation, QTL Sensor was added and incubated for 10 minutes at ~25°C. Subsequently dye-labeled phosphopeptide tracer was added with a final concentration of 0.5 μM for MBP or 125 nM for Histone H1 and PHAS-1. The plate was incubated for an additional 30 minutes at ~25°C and fluorescence measured.