IRF-1 binds to IFP35 promoter upon IFN-γ treatment.

(A) HeLa cells were either untreated or treated with IFN-γ (10 ng/ml) for 12 h. Nuclear extracts prepared from these cells were subjected to EMSA. The competitor represents 40×excess cold 3×ISRE. (B) Nuclear extracts (4 µg) from HeLa cells unstimulated or stimulated with IFN-γ (10 ng/ml) for 12 h were subjected to EMSA by using 3×ISRE probe. Supershift assays were performed by preincubating the nuclear extracts with 2 µg anti-IRF-1 or anti-IRF-3. The specific IRF-1 complex and supershifted complex were indicated by arrows. The free probes have run out of the gel. (C) HeLa cells were either untreated or treated with IFN-γ (10 ng/ml) for 12 h and processed for ChIP assays by using anti-IRF-1, anti-IRF-3 or control IgG. Precipitated DNA encompassing the IFP35 ISRE was then assayed by PCR. The negative control indicates a genomic fragment (+976 to +1337, relative to the translation start site) downstream of IL-7 promoter. (D) The experiment was similarly performed as in (C) except that anti-IRF-1 and anti-IRF-2 were used.