Figure 3

HBx directly interacts with C/EBPβ and stimulates binding of C/EBPβ to the COX-2 promoter. (A) HepG2 cells were transfected with plasmids encoding GFP-HBx or GFP alone. Cellular extracts were prepared and co-immunoprecipitated with anti-GFP antibody, and proteins were resolved by SDS-PAGE. Blots were incubated with the antibodies indicated in the figure, and were developed with chemiluminescence. (B) HepG2 cells were transfected with pG5-Luc, pRL-TK, pVP16-LAP, pM-HBx, or empty vectors, as indicated. Luciferase activity was measured in each sample, and was compared to those obtained from control cells. All data are normalized to the Renilla luciferase value, and are presented as mean ± SD, n = 3 (*P < 0.05). (C) Nuclear extracts were prepared from HepG2.2.15 and HepG2 cells, and were mixed with a ³²P-labeled C/EBP probe and analyzed by electrophoretic mobility shift assay (EMSA). Excess non-labeled C/EBP probe (100-fold) was used as a competitor. Polyclonal anti-C/EBPβ was incubated with nuclear extracts prior to addition of the probes, as indicated. (D) Nuclear extracts were prepared from HepG2 cells transfected with pCMV-HBx, and with mock transfected cells, and were mixed with a ³²P-labeled C/EBP probe and analyzed by EMSA. Excess non-labeled C/EBP probe (100-fold) was used as a competitor. (E) HepG2 cells were transfected for 48 hours with pCMV-HBx or pCMV-Tag2A, and ChIP assays were performed using 2 μg of anti-C/EBPβ. Normal rabbit IgG was used as control. Immunoprecipitated DNA or control DNA was collected and amplified using specific primers for the C/EBP binding site in the COX-2 promoter (nucleotides -155 to -2).