Elucidating Fli-1 Binding to GM-CSF Promoter

The GM-CSF promoter was screened for putative Ets DNA binding sites (EBSs) through visual inspection and utilizing the MatInspector software tool (Genomatix, Ann Arbor, MI) (35 ). Four primer pairs were designed to cover the 14 putative EBSs identified and can be found in Table 1. ChIP assay was performed using an anti-Fli-1 rabbit polyclonal antibody and normal IgG control using EpiTect ChIP OneDay Kit (Qiagen, Germantown, MD) as described (27 (link), 30 (link)). Briefly, chromatin was isolated from cross-linked HUVECs and Jurkat cells that were immunoprecipitated by antibodies specific for Fli-1 or normal rabbit IgG as a control. After immunoprecipitation, the DNA was purified and amplified by RT-PCR according to the manufacturer’s instructions (Qiagen, Germantown, MD). Fold change was calculated using the comparative Ct method 2^{− (ΔΔCT)}, where delta Ct was the difference between IgG and Fli-1 Ct values.