ChIP

To verify the direct activation of miR-92b from SP1, we performed ChIP assays with PCI-4A cells to respectively affirm the binding reaction of the 5 binding sites in miR-92b promoter region. The binding of each predicted sites were evaluated with 7 parallel groups: (A) experiment group: immunoprecipitated with SP1 antibody and amplified with targeted primers; (B) RT-Input1: without immunoprecipitation and amplified with targeted primers; (C) negative control group: immunoprecipitated with host IgG and amplified with targeted primers; (D) PCR negative control: using ddH₂O as template and amplified with targeted primers; (E) positive control group: immunoprecipitated with RNA polymerase II and amplified with GAPDH primers; (F) RT-Input2: without immunoprecipitation and amplified with GAPDH primers; (G) WB-Input: western bloting SP1 protein expression in total DNA-protein crosslink.The results indicated that there was a significant enrichment of SP1 protein at the first 3 binding sites, whereas no obvious reaction was found in site 4 and site 5.