Figure 3.

Fisetin/RSK2 complex augments its binding to YB-1 (A) Representative SPR sensorgrams showing full kinetics for direct binding characteristics between YB-1 and RSK1 (left) or YB-1 and RSK2 (right). Briefly, YB-1 was directly immobilized on the sensor chip by amine coupling and RSK1 or RSK2 was flown over the protein-coated chip at different concentrations (100-0 nM). ka and kd or steady state kinetics were used for determining the K_D. Chi square (χ²) analysis was carried out between the actual sensorgram (colored line, bottom) and the sensorgram generated from the BIAnalysis software (black line, top) to determine the accuracy of the analysis. χ² value within 1–2 was considered significant (accurate), and below 1 was highly significant (highly accurate). (B) Representative sensorgram showing full kinetics between YB-1 and RSK2. In the presence of BSA, YB-1 was directly immobilized on the sensor chip by amine coupling and RSK2 was flown over the protein-coated chip at different concentrations (50-0 nM). (C) Representative sensorgram for competition assays, where fisetin and RSK2 were flown over immobilized YB-1. The concentration of RSK2 was kept constant (100 nM), but pre-incubated with increasing amounts of fisetin (1 and 10 µM) and then injected to determine the effect of fisetin on binding activity of the RSK2 to YB-1. Data for affinity evaluation were obtained from a concentration dependent binding curve for the interaction of increasing amounts of fisetin with constant concentration of RSK2. (D) Whole cell lysates of fisetin-treated A375 melanoma cells (60 μM:24 h) with/without RSK inhibitor BI-D1870 were analyzed for YB-1 expression. Equal loading was confirmed by reprobing for vinculin.