FIG. 5.

Localization of a DNA element required for the LPS-induced enhancer activity. (A) The DNA sequence of mouse Il12b fragment C1 (−9,994 to −9,890 bp) is shown, along with the homologous sequences from the rat, human, and dog genomes. The nucleotides altered in each of the 11 substitution mutants (Sub A through Sub K) are indicated, as are three regions that are particularly well-conserved between the species. The 11 mutations were analyzed in the context of the 370-bp fragment C in the Il12b promoter-CAT reporter plasmid. Gaps introduced to maximize alignment are indicated by dashes. (B) RAW264.7 cells were transfected with enhancer-promoter-CAT reporter plasmids containing wild-type fragment C and the 11 mutant enhancers. The Il12b promoter alone (promoter) was analyzed as a control. Transfected cells were stimulated with LPS (10 μg/ml) for 24 h. (No significant activity was observed with any of the plasmids in the absence of stimulation [not shown]). The means plus standard errors of the means (error bars) from four independent experiments are shown.