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. 2015 Dec 7;211(5):1025–1040. doi: 10.1083/jcb.201505091

Figure 5.

Figure 5.

Identification of the specific DUBs responsible for NLRC5 deubiquitination. (A) The workflow showing experimental design to screen potential DUBs of NLRC5. (B) The quantified ubiquitination level of NLRC5 was regulated by USP family (related to Fig. S3 A). n = 3. The red line indicates the average NLRC5 ubiquitination level of all samples. (C) HEK293T/TLR4 cells were transfected with plasmids encoding HA-IKK-β, Flag-NLRC5, and increasing amounts of Myc-USP14, Myc-USP18, or Myc-USP22. Cell lysates were immunoprecipitated with anti-Flag antibodies followed by immunoblotting. (D) HEK293T cells were transfected with plasmids encoding HA-MyD88, Flag-NLRC5, Myc-USP14, Myc-USP18, or Myc-USP22 in the indicated combinations. Cell lysates were immunoblotted with the indicated antibodies. (E) HEK293T cells were transfected with NF-κB–luc and pRL-TK–luc reporters and the plasmids encoding MyD88, NLRC5, USP14, USP18, or USP22 in the indicated combinations and analyzed for NF-κB–dependent luciferase activity. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus the cells with MyD88 and NLRC5 overexpression (two-tailed Student’s t test). Data are representative of at least three independent experiments. n = 3. Error bars indicate the SEM. IB, immunoblotting; IP, immunoprecipitation; WCL, whole cell lysate.