Figure 8.

DCP1a inhibits IL-8 transcription and acts as a transcriptional suppressor for p65 NF-κB. (A) Stable cell lines as described in Fig. 6 and Fig. 7 were transiently transfected with the IL-8 promoter luciferase (luc.) and SV40–β-galactosidase (β-gal.) constructs. After 24 h, cells were stimulated for 4 h with 10 µg/ml IL-1α and lysed, and luciferase activity was determined. Shown is the mean luciferase activity ± SEM normalized for β-galactosidase activity from duplicate measurements of two independent experiments. (B) HEK293IL-1R cells were left untransfected or were transfected with 3 µg GFP-DCP1a, empty vector, or a GAL4 expression construct and 1 µg GAL4-p65 in the indicated combinations. 1 µg luciferase vector containing five GAL4 binding sites (pFR-luciferase) and 1 µg SV40–β-galactosidase were cotransfected. 24 h later, cells were lysed, and luciferase activity was determined. Lysates were examined for expression of GAL4 and GFP fusion proteins using anti-GAL4 or anti-GFP antibodies. Blots were probed with antiactin antibodies to confirm equal loading. Shown is the mean luciferase activity (RLU) ± SEM from six independent experiments. Numbers in percentages display relative luciferase activity of GAL4-p65 in the absence or presence of GFP-DCP1a constructs. These values were also normalized to GAL4-p65 expression levels to account for differences in GAL4-p65 amounts as in the particular Western blot shown. (C) HEK293IL-1R cells were transfected in the indicated combinations as in B but with GAL4-DCP1a constructs instead of GFP-DCP1a. Lysates were also examined for equal expression of GAL4 fusion proteins using GAL4 antibodies. A nonspecifically detected protein band served as a loading control. Shown is the mean luciferase activity (RLU) ± SEM from four independent experiments. Numbers in percentages display relative luciferase activity of GAL4-p65 in the absence or presence of GAL4-DCP1a constructs. (D) HEK293IL-1R cells were transfected with expression vectors encoding epitope-tagged p65 NF-κB or GFP-DCP1a. 24 h later, cells were lysed, and immunoprecipitations were performed using anti-p65, anti-DCP1a, or IgG control antibodies in the indicated combinations. Lysates and immunoprecipitates were analyzed by Western blotting for overexpressed p65 and DCP1a proteins as indicated. (E) HEK293IL-1R cells were treated for 1 h with 10 ng/ml IL-1α or left untreated. DCP1a and Edc4 localization was analyzed by double immune fluorescence analysis. The minus sign indicates samples from untreated cells. Bar, 10 µM. (F) Cells were treated as in D for the indicated times with 10 ng/ml IL-1α, and DCP1a and p65 proteins were analyzed in cytosolic and nuclear cell extracts by Western blotting. Black lines indicate that intervening lanes have been spliced out. mIgG, murine IgG; rIgG, rabbit IgG; IB, immunoblot; wt, wild type.