TRAF6 is required for RANKL-induced ROS production. (A) Characterization of tetracycline-regulated RANK-expressing 293 cells (293-RANK). Cell lysates (50 μg total protein) from cultures with (+) or without (–) 1 μg/mL tetracycline for 8 hours were prepared and incubated with anti-FLAG mAb. Immunoprecipitates (IPs) were analyzed by Western blotting (WB) to detect FLAG-RANK (top). 293-RANK cells induced with tetracycline were incubated for 10 minutes with (+) or without (–) RANKL (50 ng/mL), after which DCF fluorescence was measured as described in Figure 1A (bottom). (B) Effects of TRAF6.DN on RANKL-induced ROS production in 293-RANK cells. 293-RANK cells were transfected with expression constructs for TRAF6.DN (289-530)–HA as indicated. After 24 hours, the transfected cells were treated with tetracycline and further incubated for 8 hours, and then stimulated with RANKL (50 ng/mL) for 10 minutes. The production of ROSs was assayed as in Figure 1A. Immunoprecipitates were analyzed by Western blotting to detect FLAG-tagged RANKL and HA-tagged TRAF6.DN. (C) Effect of interferon-γ (IFN-γ) on ROS production. BMM cells were pretreated with 100 U/mL IFN-γ for 24 hours, and then stimulated with RANKL (50 ng/mL) for 10 minutes. The production of ROSs was assayed on the basis of DCF fluorescence as in Figure 1A. The level of TRAF6 was detected by pull-down with MBP-RANK fusion proteins (2 μg each) followed by Western blotting with polyclonal TRAF6 Ab. (D) Impairment for ROS production in splenic osteoclast precursors from TRAF6^–/– mice. Wild-type or TRAF6^–/– splenocytes were cultured in the presence of 10 ng/mL M-CSF, and then incubated for 10 minutes with (+; ▪) or without (–; ▦) RANKL (50 ng/mL). The production of ROSs was assayed as in Figure 1A. Results are representative of at least 3 independent sets of similar experiments (A-D). Data are expressed as means ± SDs of triplicates.