Fig. 3.
Overexpression of a mutant IκBα protein blocks cytokine-mediated activation and secretion of both CXCL1 and CXCL2. A and B: 832/13 cells were transfected with luciferase reporter plasmids containing −1.5 kb of CXCL1 (A) and CXCL2 (B) promoters. Posttransfection (4 h), adenoviruses expressing βGAL or IκBαSR (SR, superrepressor, which denotes S32A/S36A mutations) were transduced and cultured overnight. Cells were then untreated (open bars) or stimulated with 1 ng/ml IL-1β for 4 h (gray and filled bars). Relative promoter luciferase activity normalized to total protein is shown. *P < 0.05; **P < 0.01 vs. βGAL (gray bars). C and D: 832/13 cells were treated with indicated adenoviruses for 12 h and then stimulated with 1 ng/ml IL-1β for 6 h. Relative mRNA abundance of CXCL1 and CXCL2 was normalized to RS9. **P < 0.01 vs. βGAL (C, D). E and F: 832/13 cells were treated with indicated adenoviruses. Postadenoviral transduction (12 h), cells were treated with IL-1β for an additional 12 h. CXCL1 (E) and CXCL2 (F) release into medium was measured by ELISA and normalized to protein content via BCA assay. **P < 0.01 (E), *P < 0.05 (F). Data are shown as means ± SE from 3 individual experiments.