FIG. 2.

The NEMO[D311N] mutant form does not reconstitute IL-1-stimulated NF-κB-dependent gene transcription in NEMO-deficient cells. Wild-type NEMO (+/+) or NEMO-deficient (−/−) MEFs were cotransfected with 0.5 μg of DNA encoding an NF-κB luciferase reporter construct, 0.5 μg pTK-RL plasmid DNA (encoding Renilla luciferase), and 5 μg of empty pCMV5 vector (empty vector) or a plasmid encoding N-myc-tagged wild-type NEMO (NEMO[WT]) or NEMO[D311N] by using the Amaxa Nucleofection MEF2 kit according to the manufacturer's instructions. Subsequently, the MEFs were cultivated in Dulbecco modified Eagle medium containing 10% fetal calf serum and either left unstimulated (open bars) or stimulated (black bars) with 5 ng/ml murine IL-1α. Sixteen hours later, the cells were lysed in passive lysis buffer (Promega) and luciferase activity was measured with a dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity. The error bars show the standard deviations of duplicate experiments. To analyze protein expression, aliquots of the cell extract (10 μg protein) were immunoblotted with antibodies that recognize NEMO or GAPDH as a loading control. The positions of transfected myc-tagged NEMO and endogenous wild-type NEMO are indicated on the right.