Hcv core

Recombinant human IFN-α-2b was obtained from Schering–Plough (Kenilworth, NJ, USA). Antibodies used were: HCV core (Abcam, Cambridge, UK), NS5A (BioDesign, Saco, ME, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were peroxidase-labeled anti-mouse (GE Healthcare; Little Chalfont, UK) and Alexa Fluor 488-labeled goat anti-mouse (Invitrogen of Thermo Fisher Scientific, Waltham, MA, USA) IgG antibodies.

THP-1 cells were transfected with 100 pmol mimics-155 and negative control. The cells were lysed on ice in RIPA buffer (150 mM NaCl, 50 mM Tris-HCl, 0.5% deoxycholic acid, 0.1% SDS, 1% Triton X-100). Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membranes. Then, membranes were blocked with 5% milk-0.5% Tween-20 in Tris-buffered saline and probed with the anti-SOCS1 and anti-p-STAT1 (Cell Signaling Technology (CST), Shanghai, China) at 4 °C overnight. Subsequently, the membrane was incubated with horseradish peroxide-conjugated secondary antibody (CST). The immunoreactive bands were detected using an enhanced chemiluminescence assay kit (Beyotime Biotechnology, Shanghai, China).
THP-1 cells were divided into four groups: the control group incubated in PBS, and the experimental group treated with LPS (Sigma-Aldrich, MO), HCV CORE, or HCV NS5 (Abcam, Shanghai, China) antigens, respectively. After 1 day of culturing, the cells were lysed and protein levels were assessed by Western blot. The changes in cell signaling were detected after the stimulation of each antigen, and the phosphorylation of p65, smad, p38, and JNK proteins was detected. The applied antibodies were p65, p-smad, p-p38, and p-JNK (Abcam).