Figure 2
Various TPUF repression activity assessment in HeLa cell line. (a) Dual luciferase assay shows that TPUF (WT), a fusion of TTP (C147R) and PUM-HD (WT), exhibits the greatest down-regulation activity on FLPBS (WT) expression, compared with TTP and PUM-HD (WT) alone. Data represented as mean fold change relative to cells transfected with FLRandom (dashed line, unrepressed FL/RL activity) ± SD: n.s., not significant, ***P ≤ 0.001 (n = 3, t test). (b) Luciferase assay shows predicted specificity of previously reported PUF mutants [16]. TPUF (WT) prefers PBS (WT), TPUF (1SE) prefers PBS (A8G), and TPUF (6SE,7NQ) prefers PBS (GU/UG). Data represent means ± SD: n.s., not significant, **P ≤ 0.01, ***P ≤ 0.001 (n = 3, t test). (c) Mutations and PBSs of TPUFs A-E with 3-4 randomly chosen mutant modules. Black, WT PUF modules and corresponding RNA bases. Red, mutant PUF modules and corresponding RNA bases. Ct, C-terminus, Nt, N-terminus of the protein. (d) A graph of luciferase activity, where TPUFs A-E repress FLs with cognate PBSs. Data represent means ± SD: **P ≤ 0.01, ***P ≤ 0.001 (n = 3, t test). (e) Western blot of effector proteins using anti-Flag antibody shows no major difference in the expression. Anti-α–tubulin antibody was used as a loading control.