ChIP Assay for FOXO3a and BRD4 Binding

ChIP assays were performed as described previously^{16 (link)}. Approximately 1 × 10⁶ BT474 and T47D cells were fixed with cross-link solution and collected, ChIP assays were performed using Imprint Chromatin Immunoprecipitation Kit (Sigma, #CHP1) according to the manufacturer’s instructions. FOXO3a and BRD4 antibody-immunoprecipitated DNA was analyzed by real-time PCR. Specific primers for the CDK6 promoter were 5′-ACCTTCCCCTCCACGAGATA-3′ and 5′-GGGCGTGTGTTTAACTCCAA-3′. Unspecific primers for 3′-end region of CDK6 gene as negative control of ChIP assay were 5′-TTGGGAAAGGGAGAACTGCA-3′ and 5′-AGCACCCAGTAAGACATCCA-3′. Samples were analyzed by real-time PCR using SYBR Green Power Master Mix following the manufacturer’s protocol (Applied Biosystems). Sequential ChIP assay was performed using the Re-ChIP-IT magnetic chromatin reimmunoprecipitation kit (Active Motif, Carlsbad, CA) according to the manufacturer’s protocol. Briefly, the chromatin–IgG, chromatin–FOXO3a, or chromatin–BRD4 complex was re-immunoprecipitated using anti-BRD4, anti-FOXO3a, or anti-IgG antibodies. After the Re-ChIP assay, the isolated DNA was analyzed through quantitative RT-PCR.