FIG. 3.

Effect of the −863A polymorphism on NF-κB-dependent TNF promoter activity. (A) TNF promoter constructs (TNF −863C or TNF −863A, left panel; TNF −863C-3′UTR or TNF −863A-3′UTR, right panel) were expressed in COS-7 cells along with different ratios of CMV -p65- to CMV p50-expressing constructs. Results are shown as means and standard errors of six (left panel) and three (right panel) independent experiments. (B) Protein extracts from COS-7 cells overexpressing p65 and p50 proteins were used in an EMSA with radioactive probes corresponding to site κB1_−863C (lanes 1 through 6) or κB1_−863A (lanes 7 through 12). Lanes 2 and 8, ratio of CMV p65 to CMV p50 plasmids of 1:3; lanes 3 and 9, ratio of 1:2; lanes 4 and 10, ratio of 1:1; lanes 5 and 11, ratio of 2:1; lanes 6 and 12, ratio of 3:1. Lanes 1 and 7 show probes incubated with extracts from mock-transfected cells. The composition of the complexes was confirmed by supershift analysis (data not shown). (C) TNF −863C promoter constructs (with and without the 3′ UTR) were expressed in COS-7 cells with CMV p50 alone, with CMV p65 alone, or with a mixture of the two plasmids in a ratio of CMV p65 to CMV p50 of 3:1. RcCMV, cells transfected with an empty expression vector. Results are shown as means and standard errors of three independent experiments.