TRAF6-dependent Rac1 activation regulates RANKL-mediated ROS production and osteoclastogenesis. (A) RAW264.7 and RAW-RacN17 cells (left) or RAW264.7 cells infected with control or mutant TRAF6 (pMX-T6ΔZ3) virus (right) were treated with RANKL for 5 minutes and lysed. The lysates were incubated with GST-PBD fusion protein that binds to GTP-bound Rac1. Proteins complexed to the beads were recovered by centrifugation, and the active GTP-Rac1 and Rac1 were detected with an anti-Rac1 antibody. *P < .05. (B) RAW264.7 and RAW-RacN17 cells were treated with RANKL for 10 minutes, and ROS levels were determined as in Figure 1A. (C) RAW264.7 and RAW-RacN17 cells were treated with RANKL for 5 days. (Left) The cells were fixed and stained for TRAP, and (right) TRAP-positive MNCs were counted. Formation of TRAP-positive MNCs was severely suppressed in RacN17 cells (original magnification, × 100). Data represent means ± SDs of 3 independent experiments, each performed in duplicate (A-C).