Fig. 5.

Oligonucleotide pull-down and ChIP assay for STAT-3 binding to OIP-1 gene promoter sequence. A: Jurkat T cells stimulated with IL-12 (10 ng/ml) for 30 min in the presence/absence of anti-IFN-γ antibody. pSTAT-3 and STAT-3 expression in the total cell lysates were confirmed by Western blot analysis. Data represent three independent experiments (P <0.05). B: The nuclear extracts (NE) prepared from Jurkat cells were incubated for 2 h with biotinylated oligonucleotides containing the intact STAT binding sequence present in OIP-1 gene promoter and STAT Mut-oligo; analyzed by immunoblotting with specific anti-p-STAT-3, anti-p-STAT-1 antibody. (C) ChIP assay for STAT binding to OIP-1 promoter region. The input sample was an aliquot of nuclear extract (NE) from Jurkat T cells stimulated with IL-12 and in the presence/absence of anti-IFN-γ antibody. The input samples from Jurkat T cells were cross-linked, lysed, sonicated, and subjected to immunoprecipitation using polyclonal antibodies against pSTAT-1 and pSTAT-3. Rabbit IgG was used as a negative control antibody. The precipitated DNA fragments, input DNA, were subjected to PCR analysis using specific primers for the STAT binding region in the OIP-1 gene promoter. Data represent three independent experiments.