(A) USP25 inhibits the generation of proinflammatory cytokines in MEFs stimulated with LPS. WT or Usp25−/− MEFs were stimulated with LPS (1 μg/ml) for 3 hours before real-time reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to determine the relative abundances of the indicated mRNAs. Data are means ± SD from three independent experiments. *P < 0.01 by t test. (B and C) Deficiency in USP25 potentiates LPS-induced activation of NF-κB and MAPKs in MEFs. (B) WT or Usp25−/− MEFs were stimulated with LPS (10 μg/ml), and the degradation of IκBα and phosphorylation of ERK, JNK (c-Jun N-terminal kinase), and p38 were analyzed by Western blotting with antibodies against the indicated proteins. Data are representative of three independent experiments. *P < 0.05; **P < 0.01 by analysis of variance (ANOVA). (C) WT or Usp25
−/− MEFs were stimulated with LPS (10 μg/ml) or poly(I:C) (50 μg/ml). Nuclear extracts were prepared and subjected to EMSA analysis with NF-κB, AP-1, and Oct-1 probes. Data are representative of three independent experiments. Asterisk indicates nonspecific bands.