FIG. 6.

Effects of negatively charged residues in the C terminus of IκBα on the transcriptional and DNA-binding activities of c-Rel. (A) Effects on Rel-mediated transcription. Cos-7 cells were transfected with vector DNA or CMV plasmids encoding c-Rel or CCR.SVNLS, alone (0) or in combination with expression vectors for IκBα or IκBα mutants. Reporter plasmid pIL6CAT was included in the transfections. The normalized CAT activity from the average of three independent experiments is shown. (B) Effects on Rel DNA binding. Shown is a gel retardation analysis of whole-cell extracts (20 μg) from Cos-7 cells transfected with CMV vectors expressing c-Rel (lanes 2 to 7 and 14) or CCR.SVNLS (lanes 8 to 13), alone (lanes 2, 8, and 14) or together with vectors encoding wild-type (lanes 3 and 9) or mutant (lanes 4 to 7 and 10 to 13) IκBα proteins. Proteins were assayed for binding to a ³²P-labeled IL6-κB DNA probe. The bracket indicates the position of ectopically expressed Rel-DNA adducts. The arrowhead indicates c-Rel–DNA complexes supershifted with an anti–c-Rel antiserum (α) (lane 14). The lower band present in all lanes corresponds to a DNA-bound complex of endogenous proteins.