Simplechip plus enzymatic ip kit

CHIP analysis was performed using SimpleChIP Plus Enzymatic IP Kit (Cell Signaling Technology) according to the manufacture’s protocol. #95 THP-1-NanoLuc clone cells (4 × 10⁶ cells) were cultured with 0.1% DMSO, 1 μM BI-2536, or 1 μM BI-6727 for 24 hours and fixed in 1% formaldehyde. Cross-linked DNA fragments bound to BRD4 protein were immunoprecipitated using anti-BRD4 antibody (ab128874 [Abcam]). The amount of immunnoprecipitated DNA was determined by quantitative real-time PCR. The following primer sets were used for real-time PCR amplification of the HIV-1 LTR region: 5′-AGTGTGTGCCCGTCTGTTGT-3′, 5′-TTCGCTTTCAGGTCCCTGTT-3′. Results obtained from real-time PCR were normalized by input DNA after signals from DNA fragments imunoprecipitated with normal rabbit IgG (Cell Signaling Technology) were subtracted as a background signal.

HNF4α-transfected HEK293T cells were fixed in 1% formaldehyde for 10 min at room temperature and chromatin immunoprecipitation was carried out using SimpleChIP Plus Enzymatic IP kit (Cell Signaling Technology) and anti-HNF4α antibody (Santa Cruz Biotechnology). Purified DNA was amplified by real-time PCR using ΔΔCt method. Enrichment of the HNF4α binding site was normalized to the input samples and expressed as fold-enrichment as compared to the control normal IgG antibody. Nucleotide sequences of the primers are as follows: megalin gene with HNF4α binding site (5′-ggggttcagtaatcggaaga-3′ and 5′-gtgacaggacagcgaggtg-3′), megalin gene without HNF4α binding site (5′-ggggttcagtaatcggaaga-3′ and 5′-gtgacaggacagcgaggtg-3′), OTC gene with HNF4α binding site (5′-aaatgaggaggccaggcaa-3′ and 5′-ggttagagatactgcagggca-3′), and OTC gene without HNF4α binding site (5′-tggcaataccacactgtttagt-3′ and 5′-ctgaaccacaaggaccccaa-3′).