C ebp

Ava5 cells were seeded into a 24-well plate at a density of 5 × 10⁴ cells/well and into a 6-well plate at a density of 5 × 10⁵ cells/well. After 12–16 h of incubation, cells were treated with the reagents for appropriate duration at the indicated concentrations. Then, the cells were washed with ice-cold PBS and lysed using RIPA lysis buffer. The insoluble protein was removed by centrifugation at 12,000 rpm for 30 min at 4 °C, and the protein concentration of the soluble lysate was measured by Bio-Rad protein assay kit (Hercules, CA, USA). Immunoblotting analysis was performed as previously described. Briefly, an equal amount of protein was loaded onto 10% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The levels of the protein of interest were measured using specific antibodies against HCV NS5B (1:5000; Abcam Cambridge, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GPADH), COX-2 (1:1000; Cayman, ML, USA), Myc (1:1000; GeneTex, CA, USA), C/EBP, p-c-Jun, p-p38, t-p38, p-JNK, t-JNK, p-ERK, and t-ERK (1:1000; Cell Signaling Technology, Danvers, MA, USA). The blotting signal was developed using ECL detection kit (PerkinElmer, CT, USA) and was counted by the software Quantity One (Bio-Rad, CA, USA).

Western blotting analysis was performed according to standard protocol as we reported previously (10 (link)). Antibodies for ERK, phospho-ERK, CEBP, phospho-CEBP, CREB, phospho-CREB, REST, and β-actin were purchased from Cell Signaling.