Figure 5

IN-1 is not equally effective against all Mps1 substrates in vitro. In vitro kinase assays were performed as described by Yang et al. (2010) [28], with GST-Mps1 (0.4 mM) and either 6hisCetn2 (Cetn2) or Myelin Basic Protein (MBP) as substrate (10 mM). The IN-1 Mps1 inhibitor described by Kwiatkowski et al. (2010) [60] was included at the indicated concentrations in μM. The top and bottom panels show autoradiographs of kinase assays with Cetn2 as substrate (subst.), the bottom cropped to show just Cetn2. The middle panel shows a similar kinase assay using MBP as substrate, cropped to show just MBP. Arrows to the left indicate the signals corresponding to Mps1 autophosphorylation (which is attenuated in the presence of Cetn2), Cetn2 phosphorylation, and MBP phosphorylation. While enzyme and substrate concentrations differ from those used by Kwiatkowski et al. (2010) [60], Cetn2 phosphorylation and Mps1 autophosphorylation were observed at IN-1 concentrations that blocked MBP phosphorylation (5 and 10 μM IN-1), and residual Cetn2 phosphorylation was observed even at 100 μM IN-1.