Fig. 5. The DUB activity of USP25 is required for the regulation of TLR4-induced generation of proinflammatory cytokines and type I IFNs.

(A to C) Usp25^−/− MEFs were reconstituted with empty vector (Vec), FLAG-tagged WT USP25, or FLAG-tagged mutant USP25(C178S) (CS). (A) Reconstitution of Usp25^−/−MEFs with USP25, but not USP25(C178S), alters the LPS-induced expression of Tnf and Ifnb1. The reconstituted cells were left untreated or were stimulated with LPS (1 μg/ml) for 6 hours before real-time RT-PCR analysis of the abundances of the indicated mRNAs was performed. (B) Reconstitution of Usp25^−/− MEFs with USP25, but not USP25(C178S), inhibits LPS-induced production of IL-6 protein. The reconstituted cells were left untreated or were stimulated with LPS (1 μg/ml) for 24 hours before an ELISA assay to detect IL-6 was performed. Data are means ± SD from three independent experiments. *P < 0.05; **P < 0.01 by ANOVA. (C) LPS-induced activation of NF-κB and MAPKs is inhibited in Usp25^−/− MEFs reconstituted with USP25, but not in Usp25^−/− MEFs reconstituted with USP25(C178S). The indicated cells were left untreated or were stimulated with LPS (10 μg/ml) for 20 min before they were lysed, and the cell lysates were analyzed by Western blotting (IB) with the indicated antibodies (top five blots). Before stimulation, aliquots of cells were saved, and the abundances of the expressed proteins were examined by Western blotting with the indicated antibodies (bottom three blots). Data are representative of at least three independent experiments. The intensities of the indicated bands in (C) were quantified, and the ratios of the intensities of the corresponding bands were calculated and are shown in the bar graphs as means ± SD from three independent experiments. *P < 0.05; **P < 0.01 by ANOVA.