Deletion of TNF receptors enhances RANKL-induced activation of NF-κB. (A) Deletion of TNF receptors has no effect on the expression of RANKL signaling-related proteins. Whole-cell extracts were prepared from wild-type and TNF receptor-deleted macrophages, resolved by SDS-PAGE, and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-RANKL, TRAF1, TRAF2, TRAF6, and NIK antibodies as described in “Materials and methods.” The same membrane was reblotted with anti-β-actin antibody. (B) Time- and dose-dependent NF-κB activation by RANKL in wild-type and TNF receptor-deleted macrophages. 1.0 × 10⁶ cells were treated with 5 nM RANKL for the indicated times or with the indicated concentrations of RANKL for 30 minutes. Nuclear extracts were prepared and analyzed for NF-κB activation by EMSA as described in “Materials and methods.” (C) Graphical representation of the results shown in panel B. Results are expressed as fold activation over the untreated control; bars represent standard deviation. (D) Supershift and specificity of NF-κB. Nuclear protein was extracted from untreated or 5-nM RANKL-treated p60-^/-p80^-/- macrophages, incubated for 30 minutes with different antibodies, probed with nonlabeled NF-κB oligo, and then assayed for NF-κB activity by EMSA as described.