Crp protein

EMSAs were carried out using a LightShift chemiluminescent EMSA kit (Thermo Fisher Scientific). Biotinylated primers were used for PCR amplification of gene-specific promoters (Table S4). PCR products were concentrated and gel purified using Wizard SV Gel and PCR cleanup protocol (Promega). Binding reactions were performed with the manufacturer’s binding buffer, 100 ng poly(dI-dC), and 20 fmol of biotinylated promoter fragment in a 20-μl reaction mixture. Reaction mixtures contained increasing amounts (6.25 to 100 ng) of purified Crp protein and cAMP (Sigma) (2 mmol). Binding reactions were carried out following the manufacturer’s instructions. Nylon membranes (Hybond-N+; 30 cm) were developed using X-ray film.

The human hepatoma cell line HepG2 was purchased from Cell Resource Center, IBMS, CAMS/PUMC. HepG2 cells were cultured in DMEM (Gibco, Grand Island, NY, USA) with 10% foetal bovine serum (FBS) (Gibico), 1% NEAA (Life Technologies, Carlsbad, CA, USA), penicillin and streptomycin at 37°C in an incubator containing humidified air with 5% (v/v) CO₂, and passaged at 90% confluency with 0.25%(w/v) trypsin‐EDTA. CRP protein was obtained from Sigma‐Aldrich (St. Louis, MO, USA). HepG2 cells were treated with 10 μg/ml of CRP in vitro.