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. 2009 Apr 2;4(4):e5114. doi: 10.1371/journal.pone.0005114

Figure 5. Optineurin and E50K mutant inhibit TNFα-induced NF-κB activity.

Figure 5

(A) NF-κB-Luc reporter plasmid (25 ng) was transfected without or with optineurin expression plasmid (100 ng) in HeLa cells. After 22 hours of transfection cells were treated with TNFα for 4 hours. Luciferase activities relative to untreated control are shown (n = 3). (B) Western blot showing expression of optineurin and its mutants using HA tag antibody. Cdk2 was used as control. (C) Optineurin and E50K mutant inhibit TNFα-induced nuclear translocation of NF-κB p65. HeLa cells grown on coverslips were transfected with optineurin expression plasmid. After 24 hours of transfection cells were treated with TNFα for 30 min. The cells were then fixed and stained for optineurin (HA tag, FITC green) and p65 (Cy3, red) and visualized using a fluorescence microscope. (D) HeLa cells were infected with adenoviruses for expressing optineurin (Optn-AdV), its E50K mutant (E50K-AdV) or control virus (AdC). After 36 hours of infection, the cells were treated with TNFα for 6 min or 12 min or left untreated. Cell lysates were then prepared for western blotting with antibodies for IκBα, HA tag and Cdk2.