FIGURE 4.

TANK negatively regulates TRAF6 ubiquitination and NF-κB p65 activation, and its overexpression attenuates osteoclast differentiation. A, MDMs from wild-type Tank^−/− mice were retrovirally transfected with empty vector (LZR-CMV-IRES-GFP) or vector expressing TANK (TANK-LZR-CMV-IRES-GFP). After 3 days of culture with 25 ng/ml M-CSF plus 50 ng/ml RANKL, GFP-positive cells were identified by fluorescence microscopy (GFP) and the cells were stained for TRAP (DIC). C, mRNA levels of RANK in MDMs were measured by quantitative PCR (n = 3). D, immunoblotting analysis of anti-TRAF6 antibody immunoprecipitates from MDMs stimulated with RANKL (350 ng/ml) for indicated times probed with an anti-Ub antibody. As a loading control, immunoblotting analysis of TRAF6 was performed (bottom). E, DNA binding activity of NF-κB p65 in response to RANKL was measured using a TransAM Transcription Factor Assay Kit. Error bars: S.E. (n = 3). *, p < 0.05. F, MDMs were treated with RANKL for the indicated time and analyzed by Western blotting using anti-p100/p52 antibodies. Actin was evaluated as a loading control. The data shown are representative of three independent experiments.