FIG. 3.

Amino-terminal NEMO dimerization is sufficient for the proximity-induced IKK activation. 293 cells were transiently transfected with a luciferase reporter construct controlled by a multimerized NF-κB-binding site in conjunction with a β-actin promoter-controlled renilla luciferase reporter construct, without or in combination with a small amount (25 ng) of an IKKβ-encoding expression vector. In addition, increasing amounts of an expression vector coding for (A) the amino terminus of NEMO (amino acids 1 to 197), (B) full-length NEMO, or (C) the carboxy terminus of NEMO (amino acids 179 to 419) were added. After 24 h, the cells were lysed in TNT and the activity of the firefly as well as the renilla luciferase was determined. A mean value of the corrected relative luciferase activity of two parallel experiments is shown. This experiment was performed three times with a similar result. (D) NEMO-deficient MEFs were transiently transfected with the indicated amounts of expression vectors for FLAG-tagged IKKβ, full-length NEMO, NEMO_1-197, or NEMO_179-419 in conjunction with the NF-κB-dependent firefly luciferase reporter construct and the β-actin promoter-dependent renilla luciferase reporter construct using the Lipofectamine 2000 reagent (Invitrogen).