Figure 3

Mechanism linked to NUCKS1-mediated Tat activation. (A) HeLa cells were transfected with the V5-NUCKS1 expression plasmid. At 24 h after transfection, some of the cells were treated with PMA (50 ng/ml) for 15 min before harvest, and the cells were harvested. Nucleus and cytoplasm from the cells were fractionated. NF-κB activity was assessed by Western blotting using anti-p65, -IκB, and -NUCKS1 antibodies. The nuclear and cytoplasmic fractions were evaluated using anti-lamin B and -tubulin antibodies, respectively. (B) NUCKS1-mediated NF-κB promoter activity was assessed by luciferase assay. HeLa cells transfected with NF-κB luciferase reporter plasmid (500 ng), V5-NUCKS1 (3 μg), and pCMV-LacZ (20 ng). The luciferase assay was performed 24 h after transfection. PMA (50 ng/ml) treatment was used as a positive control. The data are expressed as mean ± SD (n = 3). (C) HeLa cells were cotransfected with the Flag-Tat and V5-NUCKS1 expression plasmids. Twenty-four hours after transfection, the nucleus and cytoplasm were fractionated, and the subcellular localization of Tat and NUCKS1 was assessed with Western blotting using anti-Flag, anti-V5, -lamin B, and -α-tubulin antibodies, respectively. (D) HeLa cells were transfected with Flag-Tat, V5-NUCKS1, or both. Two days after transfection, cell lysates were immunoprecipitated with anti-Flag antibody. Cyc T1-Tat interaction was assessed with Western blotting using anti-Cyclin T1 and anti-Flag antibodies, respectively.