Figure 8

p-STAT3/p-c-Jun/ ISL-1 forms a transcriptional complex and binds directly to ISL-1 promoter. (A) Consensus binding sites for p-STAT3 and p-c-Jun on the ISL-1 promoter were analyzed by Matinspector software. (B) The luciferase activity of ISL-1-luc was analyzed by luciferase reporter assay in Ly3 cells after treated with IL-6 (4 ng/ml), STATTIC (6 μM), Anisomycin (15 ng/ml) or SP600125 (10 μM) for 24 h. (C) ChIP assay was performed with anti-p-STAT3 Ab (left panel) or anti-p-c-Jun Ab (right panel) for immunoprecipitation using chromatin harvested from Ly3 cells. The DNA extractions were amplified using the primers that cover the p-STAT3 (primers 2) or p-c-Jun (primers 4) binding sites, or control primers (primers 1, 3) on the ISL-1 promoter by real-time PCR with normal IgG as a control. (D) Co-IP assay was performed in Ly3 for the transcriptional complex recruited on the ISL-1 promoter. (E) ChIP-re-IP assay was performed first with anti-ISL-1 Ab or rabbit IgG Ab and then with anti-p-STAT3, anti-p-c-Jun or IgG Abs for immunoprecipitation using chromatin harvested from Ly3 cells. (F) The transcriptional activity of ISL-1 on ISL-1-luc was analyzed in Ly3 cells by luciferase reporter assay. The data represent three independent experiments. Each bar represents mean ± SD. p values were calculated using a Student t-test (*p < 0.05, **p < 0.01 vs. the control).