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. 2015 Jan 1;26(1):151–160. doi: 10.1091/mbc.E14-08-1277

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© 2015 Wi, Park, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 4: — K372 ubiquitination of ECSIT is essential for the interaction and translocation of p65/p50 NF-κB into the nucleus. (A) HEK293T cells were transfected with Flag-ECSIT wt or the Flag-ECSIT K372A mutant, along with the HA-Ub vector. Immunoprecipitation (IP) with anti-Flag antibodies was performed, followed by immunoblot (IB) with antibodies to anti-Flag or anti-Ub. (B) HEK293T cells were transfected with mock, Flag-ECSIT wt, Flag-ECSIT K372A mutant, or p65- or p50-expressing vectors, as indicated. IP with anti-Flag antibody was performed, followed by IB with antibodies to anti-Flag, anti-p65, or anti-p50. (C) THP-1 cells were transfected with Flag-ECSIT wt or Flag-ECSIT K372A mutant vector and treated with LPS for the times indicated, cytosolic and nuclear fractions were isolated from cells, and a Western blot analysis was performed with antibody to anti-p65, anti-p50, anti-Flag, anti-PDI, or anti-PCNA.