Figure 3.

Mutation analysis of IL-10 promoter. A, schematic representation of putative transcription factor-binding sites in the fragment from nucleotides −265 to −1 of the human IL-10 promoter. The binding elements for transcription factors are shown in dotted boxes, and mutation sites are shown in capital letters. B, HCT 116 cells were co-transfected with the FLAG–K-RTA–expressing plasmid, internal control pRL-CMV, and mutant p-265 plasmid (as illustrated in A) (ratio 1:1:1), and luciferase assays were performed 2 days later. Basal activity of wild-type p-265 reporter plasmid without K-RTA (in empty box) was used as relative control 1. Results are presented as the mean ± S.D. (n = 3). Student's t test; *, p < 0.01 (WT versus mutant p-265). C, SP-binding site-dependent K-RTA loading onto the IL-10 promoter was determined by ABCD assay. Nuclear extracts (100 μg) from myc-K-RTA–expressing HEK293 cells were incubated with biotin-labeled IL-10 promoter probe (from either wild type or mutated mSP p-265). Myc-K-RTA co-precipitating with biotinylated DNA probes was detected by Western blotting (upper panel). Signal intensity was quantified from triplicate Western blotting experiments and calculated as p-265-bound K-RTA/input K-RTA ratio (lower plot). Student's t test; **, p < 0.05 (WT versus mSP). D, WT or mutant IL-10 promoter occupancy by K-RTA was analyzed by ChIP assay. HCT 116 cells were co-transfected with FLAG–K-RTA–expressing plasmid and p-265 (either WT or mSP) reporter plasmid (ratio 1:1) and 2 days later were fixed with 3.7% formalin. Nuclei were sonicated, and FLAG–K-RTA-bound DNA was immunoprecipitated with anti-FLAG antibody. Eluted DNA was quantified by qPCR, using primers targeting p-265. Results are presented as the mean ± S.D. (n = 3). Student's t test; *, p < 0.01.