Figure 3

Figure 3

Bucillamine inhibited afatinib-mediated EGFR kinase inhibition in vitro. (a) 10 μM afatinib was incubated with DMSO or 1 mM bucillamine for 90 min at 25 °C. The reaction products were analyzed by TLC with UV detection. (b) A recombinant EGFR kinase domain was pre-incubated with 1 or 10 nM afatinib in the presence or absence of 10–1000 μM bucillamine. Then, 10 μM ATP was added to the kinase in order to initiate phosphorylation of an artificial substrate peptide. Phosphorylation levels were measured by ELISA using antiphosphotyrosine antibody. The graph indicates the mean ± SEM (n = at least 3). DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor receptor; TLC, thin-layer chromatography; UV, ultraviolet; ATP, adenosine triphosphate; ELISA, enzyme-linked immunosorbent assay; SEM, standard error of the mean.