Figure 6

NF-κB binding domain of PIAS1 functions for N binding. (a) Schematic presentation of N and construction of N mutants. NLS, nuclear localization signal; N-N, non-covalent-interaction domain for dimerization of N; NoLS, nucleolar localization signal; β, β-sheet responsible for overall conformation of N. Numbers indicate amino acids positions. N-FL, full length N. Nuclear and nucleolar localizations, PIAS1 binding, and NF-κB activation of N and its mutants are summarized as positive (+) and negative (−). (b) NF-κB activities of N mutants. HeLa cells were transfected with plasmids containing the plasmids for NF-κB-Luciferase (0.5 μg), pRL-TK (0.05 μg), and individual mutants of N (0.5 μg) for 24 h. Cells were treated or mock-treated with TNF-α (20 ng/ml) for 6 h. Cell lysates were prepared and subjected to luciferase assays as described in Materials and Methods. Relative luciferase activities were obtained by normalizing the firefly luciferase to Renilla luciferase activities. Error bars, mean ± s.d. (n = 3). Significance was calculated by comparing to pXJ41 using two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. (c) Cellular localization of N mutants. HeLa cells were transfected with plasmids containing the N gene or each of its mutants (2 μg) of which a FLAG tag was fused at the N-terminus of each construct. At 24 h post-transfection, cells were stained with α-FLAG MAb (green) to indicate N distribution. Nuclei were stained with DAPI (blue). (d) Co-IP for HA-PIAS1 and N mutants according to description for Fig. 4b.