FIG. 7.

Activation of endogenous TAK1, IKK and JNK by RANKL. (A) TAK1 activation by RANKL in 293-RANK cells. 293-RANK cells were left unstimulated or stimulated with 1,000 ng of RANKL per ml for the indicated times. Cell extracts were immunoprecipitated with anti-TAK1 antibody, and immunoprecipitates were subjected to in vitro kinase assays using MKK6 as a substrate (upper panel). The amounts of TAK1 in each immune complex were determined by immunoblotting (lower panel). (B) Activation of IKK and JNK by RANKL. 293-RANK cells were stimulated with 1,000 ng of RANKL per ml for the indicated times. Cell extracts were immunoprecipitated with anti-IKKα or anti-JNK1 antibody. Kinase activities of IKK (top panel) and JNK (second panel) were measured by in vitro kinase assays with GST-IκB or GST-c-Jun as a substrate. Whole-cell extracts were immunoblotted with anti-IKKα and anti-JNK1 antibodies (third and bottom panels).