Supplementary Figure 1 from EMD 1214063 and EMD 1204831 Constitute a New Class of Potent and Highly Selective c-Met Inhibitors

PDF file - 45K, ATP competition studies with EMD 1214063. EMD 1214063 inhibited c-Met phosphorylation in an ATP-competitive fashion. In brief, binding competition of EMD 1214063 and ATP to c-Met was analyzed by a kinase reaction followed by Western blotting. ATP in the indicated concentrations was mixed with 1 nM EMD 1214063 and added to 100 ng recombinant human c-Met kinase (Carna Biosciences 08-151). The reaction mixtures were incubated at room temperature for 30 min. The reaction was stopped by addition of 4x LDS Sample Buffer (Invitrogen NP-0007) containing 25 mM DTT and heating at 80{degree sign}C for 20 min. The reaction mixtures were analyzed by Western blotting using 1 ng c-Met kinase per lane in gel electrophoresis. After blotting on nitrocellulose, detection was performed with an anti-phosphotyrosine antibody (clone PY99, Santa Cruz sc-7020) and an anti-GST-tag antibody (clone B-14, Santa Cruz sc-138) as a loading control. Chemiluminescence read out was measured with a VersDoc analyzer (BioRad) and band densities were quantified with the inherent software (ImageLab).