FIG. 4.

Activation of the CRE/κ3 region of the TNF-α promoter in response to LPS. (A) A single copy of the CRE/κ3 region confers LPS inducibility to a minimal TNF-α promoter. J774 cells were transfected with 2 μg of a minimal TNF-α promoter CAT reporter (−39 TNF-α CAT) CAT or reporters with one [(CRE/κ3)² −39 TNF-α CAT] or two [(CRE/κ3)¹ −39 TNF-α CAT] copies of the CRE/κ3 region. A representative CAT assay of three independent transfections is shown, illustrating CAT activity from uninduced cells and cells treated with LPS. Transfections included 2 μg of pCMVβ as a control, and CAT activity was normalized to β-Gal activity. (B) The CRE and κ3 sequences are critical for LPS inducibility of the CRE/κ3 region. J774 cells were transfected with luciferase reporters (2 μg) consisting of a minimal TNF-α promoter fused to two copies of the wild-type CRE/κ3 sequence [(CRE/κ3)² −39 TNF-α Luc] or two copies of the CRE/κ3 sequence with mutations in the CRE [(C1M)² −39 TNF-α Luc] or κ3 sequences [(3′M)² −39 TNF-α Luc]. The mutations are shown at the bottom of the figure. Histograms of luciferase activity from five independent experiments are shown; error bars indicate the standard errors of the means. All transfections included a control Renilla luciferase plasmid (2 μg), and reporter luciferase activity was normalized to Renilla luciferase activity. UN, uninduced.