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. 2015 Sep 23;5:14302. doi: 10.1038/srep14302

Figure 4. Mapping the region in 2C interacted with IKKβ.

Figure 4

(A) Full length and truncated forms of 2C inhibit NF-κB activation. 293T cells transfected with pNF-κB, pRL-TK, and 2C or 2C truncated constructs for 24 hours, were treated with TNF (10 ng/ml) for 6 hours. The cells were assayed for dual luciferase activity. Asterisks indicate significant differences between groups, data statistics were used student t-test (mean ± SD, *** indicated p < 0.001). (B) IKKβ does not interact with 2C 126-263aa. 293T cells transfected with IKKβ and 2C or 2C truncated constructs were analyzed by coimmunoprecipitation and Western blots. (C) IKKβ interacts with 2C 1-104aa and 105-125aa. 293T cells transfected with IKKβ, 2C 1-104aa, 2C 105-125aa, or GFP for 36 hours were analyzed by coimmunoprecipation and Western blot using indicated antibodies. (D) 2C 119-125aa and 122-125aa do not inhibit NF-κB activation. 293T cells transfected with pNF-κB, pRL-TK, and 2C or 2C truncated constructs for 24 hours, were treated with TNF (10 ng/ml) for 6 hours. The cells were assayed for dual luciferase activity. Asterisks indicate significant differences between groups, data statistics were used student t-test (mean ± SD, *** indicated p < 0.001). (E) 2C 122-125aa is required for binding to p65 IPT domain. IPT-GST immobilized on glutathione-Sepharose beads were incubated with lysates from 293T cell transfected with 2C truncated constructs. The bound proteins were subjected to Western blots using indicated antibodies. (F) IKKβ interacts with 2C 105-121aa. 293T cells transfected with IKKβ and 2C 1-121aa or 2C 1-125aa were analyzed by coimmunoprecipitation and Western blots.