Fig. 4: Activity and phosphorylation status of recombinant HIP kinases.
From: Abemaciclib is a potent inhibitor of DYRK1A and HIP kinases involved in transcriptional regulation
a Purification and activity measurements for all four HIPKs and DYRK1A. Recombinant protein kinases were either purified from Sf9 insect cells or from E. coli bacterial cells and analyzed by SDS-PAGE. Kinase activity was assessed using in vitro kinase assays with 0.2 mM [32P]-γ-ATP, which was incubated for 30 min either without kinase, without substrate, as a kinase-dead mutation (HIPK1D315N, HIPK2D324N, HIPK3D322N, and HIPK4D136N) or as wild-type kinase in the presence of 0.2 μM kinase and 10 μM His-c-Myc as a substrate. Measurements were performed as duplicates (n = 2 biologically independent samples) and are depicted as mean. b Molecular masses of intact kinases determined by ESI–(LC)–MS indicate the total number of phosphorylations. Source data are provided as a Source Data file.
