Effects of NDGA on the RANKL signaling pathways. RAW-D cells (a, c) or mBMMs (b) were starved in α-MEM containing 0.1% FBS for 5 h and incubated with or without NDGA for 60 min, followed by stimulation with RANKL (300 ng/ml) for indicated time periods. Total cell lysates were prepared, and equal amount of proteins were subjected to western blot analysis with the indicated Abs. (a, right panels) The density of each band was quantified by using Image J software. The vertical axis demonstrates the ratio of the expression of each protein against that of actin. (d) Luciferase assay was performed to evaluate NF-κB transcription. RAW-D cells were transiently transfected with NF-κB-dependent reporter plasmid p55IgKLuci or control vector p55Luci. Transfected cells were cultured for 24 h and then stimulated with RANKL (100 ng/ml) and varying concentrations of NDGA. The cells were then collected and the luciferase activity was measured.