Figure 2: NCI8642 is able to abolish DKK1-LRP6 interaction in vitro. (a) Kinetic analysis of the interaction of DKK1 on LRP6. DKK1 was injected at the concentrations indicated in the legend (3–54 nM), for 180 sec at the flow rate of 30 μL/min; regeneration was achieved with a very short pulse of 10 mM NaOH/1 M NaCl. A time of 500 seconds was allowed for spontaneous dissociation before regeneration step. Kinetic evaluation was achieved through the BIAevaluation software . (b) Binding of recombinant DKK1 CM injected at different dilutions over immobilized LRP6. (c) Kinetic analysis of NCI8642 on LRP6. NCI8642 was injected at the concentrations indicated in the legend (3–50 μM), for 60 sec at the flow rate of 30 μL/min. Kinetic evaluation of the small molecule was achieved through the BIAevaluation software . (d) Effect of NCI8642 on the interaction of DKK1 with LRP6. The compound was injected at a final concentrations of 5, 10, and 20 μM; DKK1 concentration was 54 nM. The two molecules were injected together after 1-hour preincubation over the immobilized LRP6. The picture is representative of two single experiments. (e) Effect of NCI8642 on the interaction of DKK1 with LRP6. When DKK1 is injected together with 20 μM NCI8642 after 1-hour incubation at 25°C, DKK1 binding on LRP6 is completely abolished (upper panel). Sequential injections of 20 μM NCI8642 and 54 nM DKK1 do not inhibit the DKK1-LRP6 interaction (middle panel). 20 μM NCI8642 is not able to displace DKK1 when injected at the end of DKK1 injection (lower panel).