Normal rabbit igg ac

Total RNA was isolated from cells or tissue using Trizol (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. QRT-PCR analysis was conducted with Applied Biosystems™ Power SYBR™ Green PCR Master Mix (ThermoFisher Scientific) using the StepOne Plus Real Time PCR System (Applied Biosystems, Foster City, CA). Gene expression levels were normalized against β-actin. In this assay, primers for human FGF19 were: 5′-GGAGATCCGCCCAGATGGCTAC-3′ (Forward) and 5′-GGCCTCCAGTCCGGTGACAAGC-3′ (Reverse). Primers for human β-actin were: 5′- GAGCACAGAGCCTCGCCTTT-3′ (Forward) and 5′-TCATCATCCATGGTGAGCTGG-3′ (Reverse). ChIP assay were performed using the anti-ATF4 antibody (Abcam) and the ChIP Assay Kit (Millipore, Burlington, MA) as described previously [7 (link)]. Normal rabbit IgG-AC (Santa Cruz Biotechnology, Dallas, TX) was used as a negative control for the antibody. The immunoprecipitated genomic DNA was amplified by qPCR using the PCR primers (Forward: 5′-ACTCCACTTTCCTCGGAATC-3′; Reverse: 5′-TCCCCCTCTTCTTCCACCGC-3′) designed to amplify a proximal promoter region containing the putative ATF4 binding element AARE (5′-CTTGATGCA-3′) on the FGF19 promoter.