Fisetin binds to RSK and suppresses its kinase activity (A) Kinase inhibitory activity: (top) Representative curve of % kinase inhibitory activity for RSK2. (bottom) % kinase inhibitory activity tabulated for RSK isoforms. Fisetin was tested for kinase activity inhibition against RSK1, RSK2 and RSK3 in 10-dose IC50 mode with 3-fold serial dilution starting at 100 μM. Reactions were carried out at 30 μM ATP. (B) Kinase binding assay: Representative curves of competition binding assay for RSK2, N and C termini. An 11-point 3-fold serial dilution of fisetin was prepared in 100% DMSO at 100X final test concentration and diluted to 1X in the assay. Kds were determined using a top concentration of 100 µM. The amount of kinase measured by qPCR (signal; y-axis) is plotted against the corresponding compound concentration in log10 scale (x-axis). (C) Binding constants for N and C termini of each RSK isoform obtained from kinase binding studies are tabulated where each experiment was performed in duplicate.