Htscan aurora b and rock2 kinase assay kit

The kinase reactions were performed as described previously ^{53 (link)}. In brief, bacterially purified recombinant PKM2 (200 ng) was incubated with MLC2 (100 ng) in kinase buffer (50 mM Tris-HCl [pH 7.5], 100 mM KCl, 50 mM MgCl₂, 1 mM Na₃VO₄, 1 mM dithiothreitol, 5% glycerol, 0.5 mM ^{32 (link)}P-PEP, and 0.05 mM fructose-1.6-bisphosphate in 25 μl at 25°C for 1 h. The reactions were terminated by adding sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and heating to 100°C.
Aurora B and ROCK2 kinase activity assays were performed using the HTScan Aurora B and ROCK2 kinase assay kit, following the manufacturer's instructions (Cell Signaling Technology, Beverly, MA).