Hcv ns5b

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).

The standard procedure of Western blotting was performed as described previously (Lee et al., 2011 (link)). The membranes were probed with monoclonal antibodies specific for HCV NS5B (1:5000; Abcam, Cambridge, MA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:10000; GeneTex, Irvine, CA, USA), anti-COX-2 antibody (1:1000; Cayman, MI, USA), anti-MAPK (phosphorylated and unphosphorylated forms of ERK1/2, p38, and JNK), anti-IKKα, anti-phospho-IKKα/β (Ser176/180), anti-NF-κB, anti-IκB-α, anti-phospho-IκB-α (Ser32) (1:1000; Cell Signaling Technology, Inc., Danvers, MA, USA), or anti-C-Myc antibody (1:1000; GeneTex, Irvine, CA, USA). The ECL detection kit was used for the signal detection (PerkinElmer, Shelton, CT, USA).

The red fluorescence protein (RFP) coding sequences were cloned in-frame at the 5′ end of DCLK1 (NM_004734) ORF to generate RFP-DCLK1 cassette in pENTR-DsRedEx2 vector (Addgene). Using clonase, the cassette was transferred to pLenti-CMV-PURO-Dest plasmid to generate pLenti-RFP-DCLK1 plasmid vector. The expression plasmid was packaged into lentivirus by transient plasmid transfection of 293T cells together with the three helper plasmids (pMD2.G, pMDL/RRE g/p, and pRSV-REV). The viral particles in the supernatants were concentrated. Following infection, the cells were selected for 7-10 days in the presence of puromycin (10 μg/ml). The resistant cells were further grown under normal culture conditions without puromycin. A control expression vector (pLenti-RFP) expresses only RFP and was packaged into viruses as described above to develop control cell lines. Total lysates were prepared from cultured cells using M-PER lysis buffer (Pierce) and Western blots were carried out by chemiluminescence method (GE Healthcare). The primary antibodies against HCV NS5B, DCLK1 and actin (all purchased from Abcam) were used for the detection of respective protein bands. The band intensities were calculated using Gelquant software to evaluate target protein to actin ratios.