FIG. 2.

TTV ORF2 protein does not alter the expression of NF-κB but affects its nuclear translocation in HeLa cells. (A) HeLa cells were transfected with the empty vector pcDNA3.1(−) as a control (lanes 1 and 3) or with pcDNA3.1(−)-TTV2 (lanes 2 and 4). At 48 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 1 h (lanes 3 and 4), cell extracts were prepared and detected by Western blot analysis with anti-p65, anti-p50, and anti-β-actin antibodies. (B) HeLa cells were transfected with 0.8 μg of either the empty vector pcDNA3.1(−) or pcDNA3.1(−)-TTV2. At 48 h posttransfection and after stimulation with TNF-α (10 ng/ml) for 1 h, cytoplasmic and nuclear fractions were prepared as described in Materials and Methods. Both cytoplasmic proteins and nuclear proteins were analyzed by SDS-PAGE and Western blotting with anti-p65 and anti-p50 antibodies to reveal the localization of NF-κB subunits. Nucleus-specific anti-YY1 antibody and anti-GAPDH antibody were used as controls. Results are representative of three different experiments.