Fig. 2. Effect of IFN- on MVP gene promoter activity and binding of STAT1. (A) Hep3B cells were transiently transfected with the indicated wild-type (pMVP1670, pMVP124) and mutated (pMVP1670-STATmut, pMVP124-STATmut) MVP promoter constructs or the vector control pGL3-Basic. Transfected cells were treated with 250 IU/ml IFN- for 6 hours prior to performing luciferase assays. Values were normalized to the protein content and are given relative to the promoter activity of untreated cells transfected with the respective promoter construct. Means and s.d. of 4 independent experiments are shown. Data were compared by Mann-Whitney U-test: *P<0.05; **P<0.01. Significant differences between untreated (dotted line) and treated cells are indicated next to the bars and between wild-type and mutated constructs next to the brackets. (B) EMSA was performed as described in the Materials and Methods using as probes a labeled oligonucleotide containing a high-affinity STAT1 site (SIE) or the wild-type (STAT-wt) and mutated (STAT-mut) GAS site of the human MVP promoter. Extracts of untreated and IFN--treated Hep3B cells were incubated with the indicated probes, unlabeled oligonucleotides (competitors) or a STAT1 antibody. ns, non-specific antibody; UK, unknown complexes. (C) ChIP assays of untreated and IFN--treated Hep3B cells were performed as described in the Materials and Methods. DNA/protein complexes were immunoprecipitated with an anti-STAT1 antibody (STAT1-Ab) or no antibody (no Ab) for control. PCR products from immunoprecipitated material were compared with those obtained from the input material (Input).