Figure 7
The role of SOCS2 in the repression of IFN signaling mediated by HBeAg. (A) HepG2 cells were transfected with pcDNA3.1 or pcDNA3.1-SOCS2 for 48 h and treated with rhIFN-α at 300 U/ml or rhIFN-λ1 at 100 ng/ml for 30 min. Cells were harvested and lysed, and p-STAT1, STAT1, SOCS2, and β-actin proteins were detected by Western blot analyses. (B) HepG2 cells were co-transfected with pISRE-Luc and pcDNA3.1 or pcDNA3.1-SOCS2 for 24 h and treated with rhIFN-α or rhIFN-λ1 for another 12 h. The activity of IFN-stimulated response element (ISRE) was measured by luciferase activity assays (upper panel). Data shown were means ± SE; n = 3. *p < 0.05. To confirm the expression of SOCS2 in pcDNA3.1-SOCS2-transfected cells, SOCS2 and β-actin proteins were detected by Western blot analyses (lower panel). (C) Determination of the efficiency of siRNA-SOCS2 in HepG2 cells. HepG2 cells were co-transfected with pCMV-tag2B or pCMV-HBeAg and the siRNA specific to SOCS2 (siR-SOCS2) or its control siRNA (siR-Ctrl) for 48 h. Cells were harvested and lysed, and SOCS2 and β-actin proteins in the cell lysates were detected by Western blot analyses. (D) HepG2 cells were co-transfected with pCMV-Tag2B or pCMV-HBeAg and siR-SOCS2 or siR-Ctrl for 48 h, and treated with rhIFN-α or rhIFN-λ1 for 30 min. (E) HepG2 cells were transfected with siR-Ctrl or siR-SOCS2 for 24 h, and incubated with rHBeAg (50 ng/ml) or PBS for another 24 h, and then treated with rhIFN-α or rhIFN-λ1for 30 min before harvest. (F) HepG2 cells were pretreated with or without PD98059 for 12 h, and incubated with rHBeAg (50 ng/ml) or PBS for 24 h, and then treated with rhIFN-α or rhIFN-λ1 for 30 min before harvest. (D–F) Cells were harvested and lysed, and p-STAT1, STAT1, β-actin, and GAPDH proteins were detected by Western blot analyses.
