FIGURE 3.
Both NF-κB and hBVR are required for TNF-α induction of GPBP. a, treatment of cells with si-hBVR prevents TNF-α induction of GPBP expression. Cells were infected with si-hBVR retrovirus 16 h before the addition of 20 ng/ml TNF-α; RNA was prepared after the indicated intervals and used as a template for random hexamer-primed cDNA synthesis. The GPBP mRNA content was determined by quantitative RT-PCR by the ΔΔCt method using 18 S rRNA as control. A representative experiment is shown; the errors were determined from the differences between the relative mRNA calculated by ΔΔCT and ΔΔCT ± S.E. (“Experimental Procedures”). b, Western blot of cells treated with siRNA for hBVR. Total cell lysates shown in a were subjected to immunoblotting. The nitrocellulose membrane was sequentially probed with anti-hBVR and anti β-actin antibodies. c, treatment of cells with si-p65 prevents TNF-α induction of GPBP expression. Cells were transfected with hBVR or with siRNA against the p65 NF-κB subunit. Eighteen hours later the cells were transferred to low serum medium and treated with 20 ng/ml TNF-α. Sample preparation and RT-PCR analysis were as in a. d, a Western blot of cells treated with siRNA for p65 is shown. Total cell lysates shown in c were subjected to immunoblotting. The nitrocellulose membrane was sequentially probed with anti-p65 and anti β-actin antibodies. e, activation of NF-κB is enhanced by elevated hBVR in the cell. HEK293A cells seeded in 6-well plates were co-transfected with the pNF-κB luciferase reporter plasmid, β-galactosidase reporter, and increasing amounts of either pEGFP-hBVR or the empty pEGFP vector as indicated. 12 h after DNA addition the cells were treated with TNF-α, and after a further 12 h the cells were harvested and lysed, and the luciferase and β-galactosidase activities were measured. Luciferase activity was normalized to that of β-galactosidase to correct for differences in transfection efficiency. f, hBVR does not alter GPBP mRNA stability. Cells were pretreated with si-hBVR retrovirus for 12 h followed by 20 ng/ml TNF-α for 6 h. The cells were then treated with 2.5 μg/ml actinomycin D (Act-D), and samples were withdrawn at the indicated times. Quantification of GPBP mRNA was as in a; the data were fitted by nonlinear regression to the first order exponential decay equation, enabling calculation of the half-life of the mRNA.