Fig. 6: CR4-WHD-tet specifically inhibits the recruitment of MKK3 to TRAF6 induced by the third treatment with RANKL.

a Coprecipitation assay using biotinylated CR4-WHD-tet. Lysates obtained from RAW264.7 cells were treated with streptavidin beads charged with or without biotinylated CR4-WHD-tet for 24 h at 4 °C. The beads and lysates were analyzed with western blotting. b Poly-ubiquitination of TRAF6. BMMs were cultured with RANKL, as shown in the schematic diagram. TRAF6 was immunoprecipitated and analyzed with western blotting using specific antibodies. c Effect of CR4-WHD-tet treatment on the activation of TAK1. BMMs were cultured with RANKL, as shown in the schematic diagram. The cell lysates were analyzed with western blotting using specific antibodies, and the intensity of each band was quantified. The activation level of TAK1 was evaluated as the amount of the phosphorylated form relative to the total protein (n = 6, mean ± SEM). *P < 0.05 (by two-sided Student’s t test). d, e Effect of CR4-WHD-tet treatment on the recruitment of MKK3 and MKK6 (d), or NEMO (e) to TRAF6. BMMs were cultured with RANKL, as shown in the schematic diagram. TRAF6 was immunoprecipitated and analyzed with western blotting using specific antibodies. The intensity of each band was quantified and presented as the amount relative to TRAF6 (n = 3 for MKK3 and NEMO, n = 4 for MKK6; mean ± SEM). *P < 0.05 (by Tukey’s test). n.s., not significant. The cells were treated with 3 μM CR4-WHD-tet 30 min before the third treatment with RANKL (b–e). f Schematic diagram of the effect of CR4-WHD-tet on the downstream signals from TRAF6.