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. 2008 Oct 28;18(11):1105–1113. doi: 10.1038/cr.2008.294

Figure 4.

Figure 4

Deubiquitinase activity was required for PLP2 to block the type I interferon signaling. (A) Enzymatic dead mutant of PLP2 DUB failed to deubiquitinate IRF3. HEK293T cell lysates overexpressing FLAG-IRF3, HA-Ubi and myc-PLP2 or myc-PLP2-C106A were immunoprecipitated with anti-FLAG antibody and ubiquitin conjugation of IRF3 was verified by immunoblotting with anti-HA antibody. The input tagged proteins were verified with indicated antibodies (bottom two panels). (B-D) PLP2-C106A became inefficient to inhibit IFNβ-luc reporter activities. IFNβ-Luc reporter assays were performed as those in Figure 2, except that pCMV-myc-PLP2-C106A was used in parallel to pCMV-myc-PLP2 (10-50 ng). Data represented the average of three independent experiments (mean±SD). (E) D277 mutation could not block the DUB activity of PLP2. Procedure was the same as in (A) except the use of pCMV-myc-PLP2-D277A. (F) PLP2-D277A could still inhibit the activation of IFNβ promoter. Procedure was the same as (B-D). Data represented the average of three independent experiments (mean±SD).