FIGURE 3.
A protein-protein interaction assay designed to screen small molecule libraries for compounds inhibiting FGF2 binding to Tec kinase. The direct interaction between FGF2 and Tec kinase was quantified using Alpha® technology (red spheres). A His-tagged form of FGF2 (62.5 nm) and GST-NΔ173Tec (31.25 nm) were used along with glutathione-coated donor and Ni-NTA-coated acceptor beads as explained under “Experimental Procedures.” As a specificity control, an unrelated protein pair, His-tagged MBP-CARP and GST-Titin (black spheres), was used. Alpha® signals were measured in the presence of increasing concentrations of untagged NΔ25FGF2, a competitor for binding of His-tagged FGF2 to GST-NΔ173 Tec. Alpha® signals are expressed as percentage of the median of the maximal Alpha® signal (tagged proteins in the absence of the NΔ25FGF2 competitor), which was set to 100% in each independent experiment. Data points represent the mean of eight independent experiments, each of which consisted of three technical replicates. Experimental deviations are expressed as S.E. (error bars). Data were fitted with a non-linear regression model. The KD of the GST-NΔ173 Tec-N25ΔFGF2 complex was calculated to be 0.63 ± 0.033 μm (S.E.) (r2 = 0.9986).