Quantitative real-time polymerase chain reaction (qRT–PCR), Western blotting, ChIP and ELISA
qRT–PCR was performed using the ABI StepOnePlusTMReal-time PCR system (Applied Biosystem) with specific primers (Table 1). The relative mRNA level of target genes was analyzed using the equation 2–ΔCt (ΔCt = Ct of the target gene – Ct of β-actin) and normalized using the level detected in the control group as 1. Western blotting experiments were performed using antibodies for FXR, LXR, and PPARγ etc., as described previously[22]. ChIP was performed as described[21]. ChIP was done using specifc antibodies for NF-κB p65 (Abcam, cat# ab16502) or PPARγ(Cat#: ab178860), and amplification of promoter sequences from the Nr1h3 (LXRα), Nr1h4 (FXR) and Bsep genes using specific primer sets and subsequent PCR; After elution and purification, the chromatin DNA was subjected to real time PCR analysis with primers, LXRα: forward: 5’- aaggaagctcaggcacaaaa -3’ and reverse: 5’- gaggctgtgcttgtgaaaca -3’, FXR: forward: 5’- ccactggagatccaaaagga -3’ and reverse: 5’- aatctatgcaaagcgctggt -3’, Bsep primer sequences: forward: 5’- tccaaattggtccacagtga -3’ and reverse: 5’- agcagcagcctcctcattac -3’. The levels of level of AST, ALT, γ-GT, and AP etc., were measured using a commercial ELISA kit (Abcam) according to the manufacturer’s protocol.