Deletion of TNF receptors enhances the RANKL-induced activation of IκBα kinase. (A) Time- and dose-dependent IKK activation by RANKL in wild-type and TNF receptor-deleted macrophages. 1.0 × 106 cells were pretreated with 50 μg/mL ALLN and then treated with 5 nM RANKL for the indicated times or with the indicated concentrations of RANKL for 15 minutes. Whole-cell extracts were prepared, incubated with anti-IKK-α antibody, and immunoprecipitated with protein A/G-Sepharose beads. The beads were washed and subjected to kinase assay as described in “Materials and methods.” Fifty micrograms of the same protein extracts was resolved by 7.5% SDS-PAGE and electrotransferred to a nitrocellulose membrane. Western blot analysis was performed using anti-IKK-α and IKK-β antibodies. (B) Graphical representation of the results shown in panel A.
