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. 2011 Mar 7;6(3):e17477. doi: 10.1371/journal.pone.0017477

Figure 4. Effect of H486R optineurin on CYLD-dependent inhibition of TNFα-induced NF-κB activity.

Figure 4

HeLa cells were transfected with optineurin or its mutants (100 ng) along with or without CYLD (100 ng, left panel and 50 ng, right panel). After 22 h of transfection, the cells were treated with TNFα for 4 h. Luciferase activities relative to untreated control are shown (n = 4). Western blot showing the expression of optineurin and its mutants along with CYLD using HA tag antibody. Yeast strain PJ694A was co-transformed with optineurin or its H486R and D474N mutants and CYLD. Transformants were grown on selection media lacking Ade to assay interaction. Growth on Ade− plate indicates interaction. GST-ubiquitin or GST alone bound to glutathione agarose beads were incubated with lysates of HEK293T cells transfected with wild type optineurin or its mutants. The bound proteins were eluted and immunoblotted with anti-HA antibodies. WCL, whole cell lysates. HeLa cells were infected with adenoviruses expressing HA-tagged wild-type or H486R mutant optineurin. After 30 hrs of infection, the cells were treated with TNFα for 5 min and immunoprecipitations were carried out with HA antibody and analyzed by Western blotting with RIP and HA antibodies.