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. 2006 Sep 25;26(24):9209–9219. doi: 10.1128/MCB.00478-06

FIG. 1.

FIG. 1.

IKK-binding domains of NEMO are weak inhibitors of TNF-α-induced NF-κB activity. (A) A schematic representation of the NEMO peptides used in the analysis. (B) 293 cells were transiently transfected with expression vectors encoding either Xpress-tagged IKKα (lanes 1 to 5) or IKKβ (lanes 6 to 10) alone or with expression vectors for FLAG-tagged IBD1 (aa 1 to 120), IBD2 (aa 40 to 120) or IBD3 (aa 60 to 120). Forty-eight hours posttransfection, whole-cell extracts were prepared and the IBD:IKK complexes were precipitated using anti-FLAG beads. The resulting samples were subjected to an immunoblot (IB) analysis using anti-Xpress and anti-FLAG antibodies as indicated. A fraction of the extracts (10%) was subjected to immunoblot analysis with the indicated antibodies to check for the expression of the various ectopically expressed proteins. (C) A similar coimmunoprecipitation experiment was performed using F-IBD1 and NEMO1-100 in conjunction with Xpress-tagged IKKα or IKKβ. (D) 293 cells were transiently transfected with 3 μg of expression vectors encoding IBD1, IBD2, or NEMO1-100. Whole-cell extracts were prepared 48 h posttransfection and were used in immunoprecipitation experiments with anti-FLAG beads. The resulting immunoprecipitates were subjected to an immunoblot analysis using either the anti-IKKα/β antibody or the FLAG antibody (left part). As a control, a fraction of the whole-cell extracts was subjected to an anti-FLAG or an anti-IKKα/β immunoblot. The anti-IKKα/β antibody preferentially recognizes the IKKβ subunit. (E) 293 cells were transiently transfected with an NF-κB-dependent firefly luciferase reporter construct (200 ng) together with a renilla luciferase construct (30 ng) without or along with the indicated amounts of the different expression vectors for IBD1, IBD2, IBD3, and NEMO1-100. Twenty-four hours posttransfection, the cells were stimulated with TNF-α for four additional hours. Subsequently the cells were harvested, whole-cell extracts were prepared, and the firefly as well as the renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean value is shown. (F) 293 cells were transiently transfected with 0 or 3 μg F-IBD1 vector prior to immunoprecipitation experiments with anti-IKKα (upper part) or anti-IKKβ antibodies (lower part). The resulting immunoprecipitates (IP) were subjected to immunoblot analysis with the indicated antibodies. NRS, normal rabbit serum.