RANKL induces eNOS activation and NO production via a TRAF6/PI3K/Akt signaling pathway. (A) HUVECs were stably transfected with a HA-tagged DN-T2 and a Flag-tagged DN-T6 using retroviral system (left). HUVECs were preincubated for 30 minutes with or without 5 μM PP1, 100 nM Wortmannin, or 1 mM NMA prior to stimulation with RANKL (5 μg/mL) for 20 minutes (right). (B) HUVECs were transiently transfected with a HA-tagged DN-Akt or a Myc-tagged Δp85. (A-B) The levels of eNOS protein and the phosphorylation of Akt and eNOS by RANKL were determined by Western blotting (top). Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of P-Akt to Akt and P-eNOS to eNOS (bottom). (C-D) eNOS activity and NO production were measured as described in Figure 3A-B. Three independent experiments were performed in duplicate. Data are means ± SDs; * *P < .01 versus RANKL alone.