Fig. 4

TRIM28 activates NF-κB signaling. (A) HEK293 cells were co-transfected with the indicated plasmids along with pNF-κB-luc plasmids or the control-luciferase plasmid and subjected to a reporter assay. The luciferase assay showed that TRIM28, but not the ΔR mutant, induced the activation of NF-κB signaling. n.s., not significant; **p < 0.01. (B) p-IκBα, IκBα, p-IKKα/β, IKKα, IKKβ, p-p65, p65, TIRM28, and β-actin detected by western blot in TRIM28-knockdown and TRIM28-overexpressed CMT-167 cells. (C) p-IκBα, IκBα, p-IKKα/β, IKKα, IKKβ, p-p65, p65, TIRM28, and β-actin detected by western blot in TRIM28-knockdown and TRIM28-overexpressed H1299 cells. (D) Western blotting analysis of IκBα expression in the indicated cells treated with TNF-α (10ng/ml). β-actin is used as a loading control. (E) Assay of NF-κB luciferase reporter gene activity in TRIM28-overexpressing CMT-167 and H1299 cells transfected with vector or the IκBα dominant negative mutant (IκBα-mu). **p < 0.01. In (A) to (C), and (E), analyses were done in triplicate. Data represent mean ± SEM from each of three independent experiments. Statistics calculated using one-way ANOVA post hoc Tukey test for multi-group or two-tailed Student’s t-test for two-group comparisons