Luciferase Reporter Assay

The putative FXR binding regions in the promoter of IL-6 were firstly predicted with online databases PROMO and NUBIscan. Then the regions were amplified by PCR from genomic DNA extracted from splenocytes in C57BL/6 WT mice and cloned using Kpn I and Sma I into the pGL-3 firefly reporter vector (Promega). All the constructs were verified by sequencing. Then respective luciferase reporter constructed or basic pGL-3 vector, as with Renilla plasmid and the FXR overexpression vector (Genecopoeia, Guangzhou, China) were co-transfected into 293T cells using lipofectamine 2000 (Thermo). Transfected cells were then lysed and luciferase activity was quantified with the Dual-Luciferase Reporter Assay (Promega) following the manufacturer’s instructions and normalized to the activity of the co-transfected Renilla reporter gene.