Fig. 5

OTUD1 removed K63-linked polyubiquitin chains of RIP2. A IP analysis of the deubiquitination of RIP2 transfected with plasmids expressing His-RIP2, HA-ubiquitin (HA-Ub) and Flag-OTUD1 (WT or C320A) in HEK293T cells. B HEK293T cells were transfected with plasmids expressing HA-Ub (WT or K48, K63), Flag-OTUD1, and His-RIP2. IP analysis of the polyubiquitination forms of RIP2 regulated by OTUD1. C Western blot analysis of RIP2 protein levels after OTUD1 overexpression in HEK 293T cells transfected with plasmids expressing Flag-OTUD1. D Western blot analysis of RIP2 in HEK 293T cells transfected with siRNA OTUD1. E Luciferase assay of NF-κB activity in HEK293T cells after transfection with control, His-RIP2 or Flag-OTUD1 (WT or C320A) vector together with the NF-κB reporter vector and Renilla luciferase vector for 36 h. Results are representative of three independent experiments, one-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05 compared with indicated group