Fig. 3.

IKKβ^SSEE induces osteoclastogenesis independent from NEMO association. (A) Bone marrow macrophages were transduced with IKKβ^SSEE in the continuous presence of NBD, mutant NBD (mNBD), or vehicle. Cells expressing IKKβ^WT were treated with RANKL in the absence of NBD or in the presence of NBD or mNBD. After 4 days, cells were stained with TRACP. Right panel shows quantification of osteoclasts per well from quadruplicate wells and four independent experiments. (B) Schematic diagram of IKKβ constructs used in panels C and D. Kinase = kinase domain; LZ = leucine zipper; HLH = helix-loop-helix; NBD = NEMO-binding domain. Not shown to scale. (C) Cells transduced with the various IKKβ forms were lysed. A portion of the lysates was used as a total cell lysate (TCL) to examine expression of IKK and NEMO proteins. Another portion of the lysate was precleared with gamma beads and subjected to immunoprecipitation using anti-Flag antibody. The coimmunoprecipitation of IKK and NEMO depicts the ability of tryptophan 739 and 741 to alanine mutations to prevent binding of IKKβ^WT and IKKβ^SSEE to NEMO (as shown in lanes annotated WA and SSEE/WA). (D) Cells transduced with the indicated IKKβ forms (shown in panel B) were plated in the absence of RANKL for 4 days. Cells then were stained with TRACP to detect osteoclasts. IKKβ^SSEE and IKKβ^SSEE/WA generated comparable numbers of osteoclasts per well (157 and 169 cells, respectively). (E) Luciferase assay for NF-κB induction by constructs shown in panel B. Cells were transduced with the indicated IKK forms, and lysates were subjected to luciferase assay, as described under “Materials and Methods.” (F) Lysates identical to those shown in panel E were subjected to IKK kinase. Expression levels of IκB, phosphorylated IκBα (p-IκBα), and IKKβ are shown.