Chip ittm kit

ChIP assay was performed by the ChIP-IT^TM kit (Active Motif, Tokyo, Japan) according to the manufacturer’s instructions. Chromatin was immunoprecipitated with rabbit LXR antibody (Santa Cruz Biotechnology Inc., Dallas, CA, USA) by using control IgG as a negative control. DNA containing the putative LXR-binding sites on rat ABCA1 promoter was amplified by PCR using forward primer 5′-GCTTTCTGCTGAGTGACTGAACTAC-3′ and reverse primer: 5′-GAATTACTGCTTTTTGCCGCG-3′ as described previously [17 (link)]. PCR was performed using TAKARA PCR thermal cycler MP in the following amplification conditions: 95 °C for 5 min, followed by 36 cycles of 95 °C for 20 s, 59 °C for 30 s, and 72 °C for 30 s. PCR products (229 bp) were detected by 3% agarose gel electrophoresis.

The chromatin immunoprecipitation (ChIP) assay was performed by the ChIP-IT^TM kit (Active Motif). Chromatin was immunoprecipitated with 2 μg of antibody of either FoxO1, TFIID, or control IgG. The harvested DNA was detected by real-time PCR and PCR to harvest the FRS fragment from −641 to −472 in the human ABCA1 promoter sequence by using sense and antisense primers 5′-AATCTCCAAGGCAGTAGGTCG-3′ and 5′-GAATCTCCCTCAGGACGCCAA-3′ [17 (link)]. PCR was performed with the TAKARA PCR thermal cycler MP in the amplification program: 95 °C for 5 min, followed by 36 cycles of 95 °C for 20 s, 61 °C for 30 s, and 72 °C for 30 s. The products of the PCR (169 bp) were detected by 3% agarose gel electrophoresis.