Fig. 5. Effect of myricetin on UVB-mediated Raf kinase activity and binding activity in mouse skin.

(A) Myricetin inhibits UVB-induced Raf kinase activity. Mice were treated as described for Figure 1B. Raf kinase activity was determined using immunoprecipitation followed by a kinase assay as described in “Materials and methods”. Results are shown as means ± S.E. (n = 5). The symbol (#) indicates a significant difference (p < 0.05) between the control group and the UVB-irradiated group. Asterisks (*) indicate a significant difference (p < 0.05) between the myricetin-treated and non-treated groups of irradiated mice. (B) Myricetin directly binds Raf in mouse skin lysates. Mice were treated as described for Figure 1B. In vivo myricetin binding was confirmed by Western blot using an antibody against Raf: lane 1 (input control), whole lysate from mouse dorsal skin; lane 2 (control), mouse dorsal skin lysate precipitated with Sepharose 4B beads; and lane 3, whole-cell lysate from mouse dorsal skin precipitated by myricetin-Sepharose 4B affinity beads. (C) Myricetin binds Raf directly in an ATP-noncompetitive manner. Active Raf (2 g) was incubated with ATP at different concentrations (10 or 100 M) and 50 l of myricetin–Sepharose 4B or 50 l of Sepharose 4B (as a negative control) in reaction buffer at a final volume of 500 l. The mixtures were incubated at 4°C overnight with shaking. After washing, the pulled-down proteins were analyzed by Western blot: lane 2, negative control, Raf cannot bind with Sepharose 4B; lane 3, positive control, Raf binding with myricetin–Sepharose 4B; lanes 4 and 5, increasing amounts of ATP did not suppress myricetin binding with Raf. Each experiment was performed 3 times.